Pan-cancer analysis of mutation density at constitutive origins
Pierre Murat
07 October, 2024
This notebook reports the analysis of mutational burden (somatic snvs/indels and SVs) at constitutive origins.
1 Data formating
1.1 Simple somatic mutations
Format aggregated simple somatic mutations ICGC data
Simple somatic mutations are from the International Cancer Genome Consortium (ICGC) - Release 28 (https://dcc.icgc.org/releases/release_28). Data is using GRCh37/hg19 coordinates.
# Convert vcf file to bed file reporting the position and nature of variants together with the variant frequency
# with perl/bash
perl -lane 'print join "\t",(@F[0,1,3,4], /(?:OCCURRENCE)=[^;]+/g)' /cephfs/pmurat/OriVir/Dataset/ICGC/simple_somatic_mutation.aggregated.vcf \
> /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/simple_somatic_mutation.aggregated.tsv
# Reformat
sed 's/\OCCURRENCE=//g' /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/simple_somatic_mutation.aggregated.tsv | \
awk '{split ($5, T, "|"); $5 = T[1] OFS T[3] OFS T[4]}1' OFS="\t" | sed 's/,.*$//' \
> /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/simple_somatic_mutation.aggregated.2.tsv
# Remove comment lines
grep -o '^[^#]*' /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/simple_somatic_mutation.aggregated.2.tsv \
> /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.simple.somatic.mutation.aggregated.tsv
# delete temporary files
rm -f /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/simple_somatic_mutation.aggregated.tsv
rm -f /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/simple_somatic_mutation.aggregated.2.tsv
# count variants
wc -l /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.simple.somatic.mutation.aggregated.tsv
# 81,782,588 variants
# Split snvs and indels
awk 'length($3)==1 && length($4)==1' /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.simple.somatic.mutation.aggregated.tsv \
> /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.tsv
awk 'length($3)!=1 || length($4)!=1' /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.simple.somatic.mutation.aggregated.tsv \
> /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.tsv
# count variants
wc -l /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.tsv
# 74,817,293 snvs
wc -l /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.tsv
# 6,965,295 indels
# Sort files
sort -k1,1 -k2,2n /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.tsv > /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.sorted.tsv
sort -k1,1 -k2,2n /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.tsv > /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.sorted.tsv
# Duplicate second column and generate bed files
# snvs
awk '{print $1,$2,$2+=1,$3,$4,$5,$6,$7}' /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.sorted.tsv > /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.temp.bed
cat /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.temp.bed | tr ' ' '\t' > /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.sorted.bed
rm -f /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.temp.bed
# indels
awk '{print $1,$2,$2+=1,$3,$4,$5,$6,$7}' /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.sorted.tsv > /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.temp.bed
cat /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.temp.bed | tr ' ' '\t' > /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.sorted.bed
rm -f /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.temp.bed
1.2 Structural somatic mutations
Generated manifest from the ICGC data portal using the following filtering criteria: StSM > Collaboratory - Toronto > Broad variant call pipeline –> 5,830 files from 1,830 donors (manifest.1698320954196.tar.gz)
Load and modify the manifest
# r
library(dplyr)
library(stringr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
#
SV.manifest.df <- read.table(gzfile("./Dataset/ICGC/SV/manifest.1698320954196.tar.gz"), skip = 1)
SV.manifest.df <- SV.manifest.df %>% dplyr::select(V9, V10, V2, V3, V5)
colnames(SV.manifest.df) <- c("donor.id", "project", "file.id", "object.id", "file.name")
# Select only SV detected by both snowman and dRanger
SV.manifest.snowman.dRanger.df <- SV.manifest.df %>% filter(str_detect(file.name, 'dRanger_snowman')) # 1,940 entries, 1,820 donors
# Save manifest
write.table(SV.manifest.snowman.dRanger.df, "./Dataset/ICGC/SV/SV.manifest.tsv", sep="\t", col.names = F, row.names = F, quote = F)Download files with score-client using the /cephfs2/pmurat/OriCan/Scripts/sv.score.client.sh
Aggregate data
#
library(tidyr)
# Using a loop on the previous manifest table
# List vcf files
vcf.files <- list.files("./Dataset/ICGC/SV/VCF/")
vcf.files <- vcf.files[!grepl(".idx", vcf.files)]
#
SV.aggregate.df <- tibble()
for (i in 1:length(vcf.files)) {
vcf.files.i <- vcf.files[i]
print(vcf.files.i)
df.i <- SV.manifest.snowman.dRanger.df %>% filter(file.name == vcf.files.i)
donor.i <- df.i$donor.id
project.i <- df.i$project
path.i <- paste("/Volumes/cephfs2/pmurat/OriCan/Dataset/ICGC/SV/VCF/", vcf.files.i, sep = "")
vcf.i <- read.table(gzfile(path.i), comment.char = "#") %>% dplyr::select(V1, V2, V3, V4, V5, V8)
vcf.i <- vcf.i %>% mutate(V9 = str_c(str_match(V8, ";SVCLASS=(.*);")[, 2])) %>% dplyr::select(-V8) %>% mutate(V10 = donor.i, V11 = project.i)
vcf.i[is.na(vcf.i)] <- "n_d"
SV.aggregate.df <- rbind(SV.aggregate.df,vcf.i)
}
write.table(SV.aggregate.df, "./Dataset/ICGC/SV/ICGC.somatic.SV.aggregated.tsv", sep="\t", col.names = F, row.names = F, quote = F)
nrow(SV.aggregate.df) # 1,457,904 structural variants# Sort file
sort -k1,1 -k2,2n /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.somatic.SV.aggregated.tsv > /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.somatic.SV.aggregated.sorted.tsv
# Duplicate second column to generate a bed file
awk '{print $1,$2,$2+=1,$3,$4,$5,$6,$7,$8}' /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.somatic.SV.aggregated.sorted.tsv > /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.SV.temp.bed
cat /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.SV.temp.bed | tr ' ' '\t' > /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.SV.aggregated.sorted.bed
rm -f /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.SV.temp.bed1.3 Replication origins
1.3.1 Formatting
Format origin coordinates and compute origin centers
# r
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load origin coordinates
constitutive.ori.hg38.df <- read.table("./Dataset/ori/GSM5658908_Ini-seq2.called.replication.origins.bed", skip=1)
# Add origin id
constitutive.ori.hg38.df <- constitutive.ori.hg38.df %>% mutate(ori.id = paste("ori", c(1:nrow(constitutive.ori.center.hg38.df)), sep = "."))
# Compute origin width
constitutive.ori.hg38.df <- constitutive.ori.hg38.df %>% mutate(ori.width = V3-V2)
# Compute origin center
constitutive.ori.center.hg38.df <- constitutive.ori.hg38.df %>% mutate(V2 = round((V2+V3)/2), V3=V2+1)
# Save
write.table(constitutive.ori.center.hg38.df, "./Dataset/ori/ori.center.hg38.bed", sep="\t", col.names = F, row.names = F, quote = F)Liftover origin coordinates to hg19
# Liftover to hg19
liftOver -bedPlus=3 /cephfs2/pmurat/OriCan/Dataset/ori/ori.center.hg38.bed /cephfs/pmurat/Genomes/LiftOver_chains/hg38ToHg19.over.chain.gz /cephfs2/pmurat/OriCan/Dataset/ori/ori.center.hg19.bed /cephfs2/pmurat/OriCan/Dataset/ori/ori.liftover.unmapped
# Remove "chr"
sed 's/^chr\|%$//g' /cephfs2/pmurat/OriCan/Dataset/ori/ori.center.hg19.bed > /cephfs2/pmurat/OriCan/Dataset/ori/ori.center.hg19.NCBI.bed
# Sort
sort -k1,1 -k2,2n /cephfs2/pmurat/OriCan/Dataset/ori/ori.center.hg19.NCBI.bed > /cephfs2/pmurat/OriCan/Dataset/ori/ori.center.hg19.NCBI.sorted.bedSummary - 23,838 constitutive origins
1.3.2 Filtering
We exclude origin clusters to avoid complex/overlapping signals. To so, we filter out origins with an inter-origins distance smaller than 20kb.
# r
# Constitutive origin filtering
ori.center.df <- read.table("./Dataset/ori/ori.center.hg19.NCBI.sorted.bed")
ori.inter.dist.df <- ori.center.df %>% mutate(prev = V2-lag(V2), nxt = lead(V2)-V2) %>%
mutate(min = pmin(prev, nxt, na.rm = T)) %>% mutate(min = ifelse(min > 0, min, NA)) %>%
filter(min >= 20000) %>% dplyr::select(-prev, -nxt, -min)
# 9,341 remaining origins
# Save final origin coordinate bed files
write.table(ori.inter.dist.df, "./Dataset/ori/ori.inter.hg19.NCBI.bed", sep="\t", col.names = F, row.names = F, quote = F)Summary - 9,341 selected constitutive origins
1.3.3 Reshuffle coordinates
In order to compare mutation rates to basal level, origin coordinates are reshuffled using bedtools reshuffle. A hg19.genome file is prepared from the fai file of hg19.
# bash
bedtools shuffle -i /cephfs2/pmurat/OriCan/Dataset/ori/ori.inter.hg19.NCBI.bed \
-g /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/hg19.genome.fai -chrom \
> /cephfs2/pmurat/OriCan/Dataset/ori/ori.reshuffle.hg19.NCBI.bed
sort -k1,1 -k2,2n /cephfs2/pmurat/OriCan/Dataset/ori/ori.reshuffle.hg19.NCBI.bed > /cephfs2/pmurat/OriCan/Dataset/ori/ori.reshuffle.hg19.NCBI.sorted.bed 1.3.4 Origin base composition
Compute base composition of selected origins for correcting mutation rates for local sequence composition effects
1.3.4.1 Base composition statistics
Select 10 kb domains centered on origins
# r
ori.10kb.df <- ori.inter.dist.df %>% mutate(V2 = V2-10000, V3 = V3+9999)
write.table(ori.10kb.df, "./Dataset/ori/ori.10kb.domain.hg19.NCBI.bed", sep="\t", col.names = F, row.names = F, quote = F)Partition origin domains in 100 nucleotides bins
# bash
bedtools makewindows -b /cephfs2/pmurat/OriCan/Dataset/ori/ori.10kb.domain.hg19.NCBI.bed \
-w 100 -i winnum > /cephfs2/pmurat/OriCan/Dataset/ori/ori.10kb.domain.100nt.split.hg19.NCBI.bedCompute base composition
# r
library(rtracklayer)
library(BSgenome.Hsapiens.UCSC.hg19)
# Load domain/bin coordinates
ori.10kb.domain.100nt.split.gr <- import("./Dataset/ori/ori.10kb.domain.100nt.split.hg19.NCBI.bed")
my.chr <- c(1:22, "X", "Y")
ori.10kb.domain.100nt.split.gr <- ori.10kb.domain.100nt.split.gr[seqnames(ori.10kb.domain.100nt.split.gr) %in% my.chr]
seqlevelsStyle(ori.10kb.domain.100nt.split.gr) <- "UCSC"
# Recover sequences and compute base composition statistics
ori.views <- Views(Hsapiens, ori.10kb.domain.100nt.split.gr)
ori.bc <- letterFrequency(ori.views, c("A", "C", "G", "T"), as.prob = FALSE)
ori.bc.df <- cbind.data.frame(bin = ori.10kb.domain.100nt.split.gr$name, ori.bc)
ori.bc.summary <- ori.bc.df %>% group_by(bin) %>% summarise_at(c("A", "C", "G", "T"), sum) %>%
mutate(dist = (as.numeric(bin)-100)*100,
A.freq = A/(A+C+T+G),
C.freq = C/(A+C+T+G),
G.freq = G/(A+C+T+G),
T.freq = T/(A+C+T+G),
GC=(G+C)/(A+C+T+G))
write.csv(ori.bc.summary, "./Dataset/ori/ori.bc.summary.csv", quote = F, row.names = F)1.3.4.2 Base composition of individual origins
Select 10 kb domains centered on origins
# r
ori.1kb.df <- ori.inter.dist.df %>% mutate(V2 = V2-500, V3 = V3+499)
write.table(ori.1kb.df, "./Dataset/ori/ori.1kb.domain.hg19.NCBI.bed", sep="\t", col.names = F, row.names = F, quote = F)Compute base composition of individual origins
# r
library(rtracklayer)
library(BSgenome.Hsapiens.UCSC.hg19)
# Load domain/bin coordinates
ori.1kb.domain.bed <- read.table("./Dataset/ori/ori.1kb.domain.hg19.NCBI.bed")
colnames(ori.1kb.domain.bed) <- c("seqnames", "start", "end", "EFF", "ori.id")
ori.1kb.domain.gr <- makeGRangesFromDataFrame(ori.1kb.domain.bed, keep.extra.columns = T)
my.chr <- c(1:22, "X", "Y")
ori.1kb.domain.gr <- ori.1kb.domain.gr[seqnames(ori.1kb.domain.gr) %in% my.chr]
seqlevelsStyle(ori.1kb.domain.gr) <- "UCSC"
# Recover sequences and compute base composition statistics
ori.1kb.views <- Views(Hsapiens, ori.1kb.domain.gr)
ori.1kb.bc <- letterFrequency(ori.1kb.views, c("A", "C", "G", "T"), as.prob = FALSE)
ori.1kb.bc.df <- cbind.data.frame(ori.id = ori.1kb.domain.gr$ori.id, EFF = ori.1kb.domain.gr$EFF, ori.1kb.bc)
write.csv(ori.1kb.bc.df, "./Dataset/ori/ori.1kb.bc.summary.csv", quote = F, row.names = F)2 Mutational burden
2.1 Origin mutation density
Compute distance from mutations to the closest origin (bedtools closest)
# bash
# at origins
# snvs
bedtools closest -a /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.sorted.bed \
-b /cephfs2/pmurat/OriCan/Dataset/ori/ori.inter.hg19.NCBI.bed -D ref \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.bed
# indels
bedtools closest -a /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.sorted.bed \
-b /cephfs2/pmurat/OriCan/Dataset/ori/ori.inter.hg19.NCBI.bed -D ref \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.bed
# SVs
bedtools closest -a /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.SV.aggregated.sorted.bed \
-b /cephfs2/pmurat/OriCan/Dataset/ori/ori.inter.hg19.NCBI.bed -D ref \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.bed
# at reshuffled origins
# snvs
bedtools closest -a /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.sorted.bed \
-b /cephfs2/pmurat/OriCan/Dataset/ori/ori.reshuffle.hg19.NCBI.sorted.bed -D ref \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.reshuffle.bed
# indels
bedtools closest -a /cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/ICGC.indels.aggregated.sorted.bed \
-b /cephfs2/pmurat/OriCan/Dataset/ori/ori.reshuffle.hg19.NCBI.sorted.bed -D ref \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.reshuffle.bed
# SVs
bedtools closest -a /cephfs2/pmurat/OriCan/Dataset/ICGC/SV/ICGC.SV.aggregated.sorted.bed \
-b /cephfs2/pmurat/OriCan/Dataset/ori/ori.reshuffle.hg19.NCBI.sorted.bed -D ref \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.reshuffle.bedSelect mutations in proximity to origins (+- 10kb)
# origins
# snvs
awk '$14>=-10000 && $14<=10000' /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.bed \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.bed
# indels
awk '$14>=-10000 && $14<=10000' /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.bed \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.10kb.bed
# SVs
awk '$15>=-10000 && $15<=10000' /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.bed \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.10kb.bed
# reshuffled origins
# snvs
awk '$14>=-10000 && $14<=10000' /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.reshuffle.bed \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.reshuffle.bed
# indels
awk '$14>=-10000 && $14<=10000' /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.reshuffle.bed \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.10kb.reshuffle.bed
# SVs
awk '$15>=-10000 && $15<=10000' /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.reshuffle.bed \
> /cephfs2/pmurat/OriCan/001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.10kb.reshuffle.bedCompute raw mutation count at origins using the reshuffled coordinates for assessing background.
Distances computed by bedtools are inverted to phase the analysis on origins.
# r
# Load data
# snvs
ICGC.snvs.closest.ori.10kb.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.bed")
ICGC.snvs.closest.ori.10kb.reshuffle.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.reshuffle.bed")
# indels
ICGC.indels.closest.ori.10kb.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.10kb.bed")
ICGC.indels.closest.ori.10kb.reshuffle.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.indels.closest.ori.10kb.reshuffle.bed")
# SVs
ICGC.SV.closest.ori.10kb.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.10kb.bed")
ICGC.SV.closest.ori.10kb.reshuffle.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.SV.closest.ori.10kb.reshuffle.bed")
# Compute count in 100 nt windows
# snvs
ICGC.snvs.ori.count.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(V14 = -V14) %>% mutate(dist = (as.numeric(cut(V14, breaks = seq(-10000, 10000, 100)))-100)*100) %>% group_by(dist) %>% summarise(snvs.count=n())
ICGC.snvs.ori.count.reshuffle.df <- ICGC.snvs.closest.ori.10kb.reshuffle.df %>% mutate(V14 = -V14) %>% mutate(dist = (as.numeric(cut(V14, breaks = seq(-10000, 10000, 100)))-100)*100) %>% group_by(dist) %>% summarise(snvs.count.reshuffle=n())
# indels
ICGC.indels.ori.count.df <- ICGC.indels.closest.ori.10kb.df %>% mutate(V14 = -V14) %>% mutate(dist = (as.numeric(cut(V14, breaks = seq(-10000, 10000, 100)))-100)*100) %>% group_by(dist) %>% summarise(indels.count=n())
ICGC.indels.ori.count.reshuffle.df <- ICGC.indels.closest.ori.10kb.reshuffle.df %>% mutate(V14 = -V14) %>% mutate(dist = (as.numeric(cut(V14, breaks = seq(-10000, 10000, 100)))-100)*100) %>% group_by(dist) %>% summarise(indels.count.reshuffle=n())
# SVs
ICGC.SV.ori.count.df <- ICGC.SV.closest.ori.10kb.df %>% mutate(V15 = -V15) %>% mutate(dist = (as.numeric(cut(V15, breaks = seq(-10000, 10000, 100)))-100)*100) %>% group_by(dist) %>% summarise(SV.count=n())
ICGC.SV.ori.count.reshuffle.df <- ICGC.SV.closest.ori.10kb.reshuffle.df %>% mutate(V15 = -V15) %>% mutate(dist = (as.numeric(cut(V15, breaks = seq(-10000, 10000, 100)))-100)*100) %>% group_by(dist) %>% summarise(SV.count.reshuffle=n())
# Join and save results
ICGC.mut.count.df <- ICGC.snvs.ori.count.df %>% left_join(ICGC.snvs.ori.count.reshuffle.df) %>% left_join(ICGC.indels.ori.count.df) %>% left_join(ICGC.indels.ori.count.reshuffle.df) %>% left_join(ICGC.SV.ori.count.df) %>% left_join(ICGC.SV.ori.count.reshuffle.df)
saveRDS(ICGC.mut.count.df, "./001_Mut_density_Pan_cancer/rds/ICGC.mut.count.df.rds")Plot
library(dplyr)
library(tidyr)
library(ggplot2)
library(ggpubr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load df
ICGC.mut.count.df <- readRDS("./001_Mut_density_Pan_cancer/rds/ICGC.mut.count.df.rds")
# Plot snvs
# snvs
ICGC.snvs.count.plot <- ICGC.mut.count.df %>% dplyr::select(dist, snvs.count, snvs.count.reshuffle) %>% gather(variable, value, -dist) %>% ggplot(aes(x=dist, y=value, colour=variable)) + geom_line() +
scale_color_manual(values = c("#E41F1A", "#4F4A75")) +
xlim(-5000,5000) + ylim(15000,32000) +
xlab("Distance from origin (bp)") + ylab("snvs count") + ggtitle("snvs distribution at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.snvs.count.plot
# indels
ICGC.indels.count.plot <- ICGC.mut.count.df %>% dplyr::select(dist, indels.count, indels.count.reshuffle) %>% gather(variable, value, -dist) %>%
ggplot(aes(x=dist, y=value, colour=variable)) + geom_line() +
scale_color_manual(values = c("#E41F1A", "#4F4A75")) +
xlim(-5000,5000) + ylim(1700,3500) +
xlab("Distance from origin (bp)") + ylab("indels count") + ggtitle("indels distribution at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.indels.count.plot
# SVs
ICGC.SV.count.plot <- ICGC.mut.count.df %>% dplyr::select(dist, SV.count, SV.count.reshuffle) %>% gather(variable, value, -dist) %>%
ggplot(aes(x=dist, y=value, colour=variable)) + geom_line() +
scale_color_manual(values = c("#E41F1A", "#4F4A75")) +
xlim(-5000,5000) + ylim(300,700) +
xlab("Distance from origin (bp)") + ylab("SV count") + ggtitle("SV distribution at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.SV.count.plot
pdf("./Rplots/ICGC.snvs.count.plot.pdf", width=7, height=6, useDingbats=FALSE)
ICGC.snvs.count.plot
dev.off()## quartz_off_screen
## 2pdf("./Rplots/ICGC.indels.count.plot.pdf", width=7, height=6, useDingbats=FALSE)
ICGC.indels.count.plot
dev.off()## quartz_off_screen
## 2pdf("./Rplots/ICGC.SV.count.plot.pdf", width=7, height=6, useDingbats=FALSE)
ICGC.SV.count.plot
dev.off()## quartz_off_screen
## 2# Compute fold enrichment
ICGC.mut.ori.FC.df <- ICGC.mut.count.df %>% mutate(class = case_when(dist == 0 ~ "ori",
dist <= -1000 | dist >= 1000 ~ "flanks",
T ~ "other")) %>% group_by(class) %>%
summarise(snvs.mean = mean(snvs.count, na.rm = T), indels.mean = mean(indels.count, na.rm = T), SV.mean = mean(SV.count, na.rm = T))
# snvs: 30685/18008 = 1.703965
# indels: 30685/18008 = 1.078405
# SVs: 30685/18008 = 1.5034012.2 Origin clustering
Assess mutational burden at origin cluster
# R
# Load origin coordinates
ori.center.df <- read.table("./Dataset/ori/ori.center.hg19.NCBI.sorted.bed")
colnames(ori.center.df) <- c("seqnames", "start", "end", "EFF", "ori.id", "ori.width")
ori.df <- ori.center.df %>% mutate(start = round(start-(ori.width/2)), end = round(start+(ori.width/2)))
ori.gr <- makeGRangesFromDataFrame(ori.df, keep.extra.columns = T)
seqlevelsStyle(ori.gr) <- "UCSC"
# 23,838 origins
# Merge intervals that are at less than 20 kb
ori.merge.10k.gr <- reduce(ori.gr, min.gapwidth=20000L, , ignore.strand=TRUE)
summary(width(ori.merge.10k.gr))
hist(log10(width(ori.merge.10k.gr)), breaks = 100)
# Define origin clusters
ori.cluster.gr <- ori.merge.10k.gr[width(ori.merge.10k.gr) >= 20000]
hist(log10(width(ori.cluster.gr)), breaks = 100)
ori.cluster.gr$cluster.id <- paste0("cluster.", seq(1, length(ori.cluster.gr), 1))
summary(width(ori.cluster.gr))
# Min. 1st Qu. Median Mean 3rd Qu. Max.
# 20001 26601 36401 52027 59451 553601
# Compute the number of origins per cluster
ori.cluster.count.df <- as.data.frame(mergeByOverlaps(ori.cluster.gr, ori.gr)) %>% group_by(cluster.id) %>% summarise(ori.count = n())
summary(ori.cluster.count.df$ori.count)
# Min. 1st Qu. Median Mean 3rd Qu. Max.
# 2.000 4.000 5.000 7.538 9.000 118.000
# Prepare and save bed cluster bed file
seqlevelsStyle(ori.cluster.gr) <- "NCBI"
ori.cluster.count.bed <- as.data.frame(ori.cluster.gr) %>% select(seqnames, start, end)
write.table(ori.cluster.count.bed, "./Dataset/ori/ori.cluster.bed", sep="\t", col.names = F, row.names = F, quote = F)Recover mutations in the vicinity of origin clusters
# bash
sort -k1,1 -k2,2n /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/ori.cluster.bed > /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/ori.cluster.sorted.bed
/Users/pm23/Desktop/Software/bedtools2/bin/bedtools closest \
-a /Users/pm23/Desktop/Projects/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.sorted.bed \
-b /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/ori.cluster.sorted.bed -D ref \
> /Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.cluster.bed
# Select mutations in proximity to origins (+- 10kb)
awk '$12>=-50000 && $12<=50000' /Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.cluster.bed \
> /Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.cluster.50kb.bedProcess
# R
# Load resulting df
ICGC.snvs.closest.ori.cluster.50kb.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.cluster.50kb.bed") %>% select(V1, V2, V3, V4, V5)
colnames(ICGC.snvs.closest.ori.cluster.50kb.df) <- c("seqnames", "start", "end", "REF", "ALT")
ICGC.snvs.closest.ori.cluster.50kb.gr <- makeGRangesFromDataFrame(ICGC.snvs.closest.ori.cluster.50kb.df, keep.extra.columns = T)
ICGC.snvs.closest.ori.cluster.50kb.gr$score <- 1
# Compute the distribution of snvs at origin cluster
snvs.ori.cluster.mat <- normalizeToMatrix(ICGC.snvs.closest.ori.cluster.50kb.gr, ori.cluster.gr, value_column = "score", mean_mode = "coverage", extend = 50000, w = 1000, target_ratio = 0.5, background = 0)
saveRDS(snvs.ori.cluster.mat, "./001_Mut_density_Pan_cancer/rds/snvs.ori.cluster.mat.rds"Compare the number of snvs at origin within clusters or isolated
# R
ori.center.df <- read.table("/Users/pm23/Desktop/Projects/OriCan/Dataset/ori/ori.center.hg19.NCBI.sorted.bed")
ori.isol.df <- ori.center.df %>% mutate(prev = V2-lag(V2), nxt = lead(V2)-V2) %>%
mutate(min = pmin(prev, nxt, na.rm = T)) %>% mutate(min = ifelse(min > 0, min, NA)) %>%
filter(min >= 20000) %>% dplyr::select(-prev, -nxt, -min)
colnames(ori.isol.df) <- c("seqnames", "start", "end", "EFF", "ori.id", "ori.width")
ori.isol.gr <- makeGRangesFromDataFrame(ori.isol.df, keep.extra.columns = T)+500
# 9341 origins
ori.cluster.df <- ori.center.df %>% mutate(prev = V2-lag(V2), nxt = lead(V2)-V2) %>%
mutate(min = pmin(prev, nxt, na.rm = T)) %>% mutate(min = ifelse(min > 0, min, NA)) %>%
filter(min <= 10000) %>% dplyr::select(-prev, -nxt, -min)
colnames(ori.cluster.df) <- c("seqnames", "start", "end", "EFF", "ori.id", "ori.width")
ori.cluster.gr <- makeGRangesFromDataFrame(ori.cluster.df, keep.extra.columns = T)+500
# 11197 origins
# Load snvs coordinates for cluster and isolated origins
ICGC.snvs.closest.ori.10kb.df <- read.table("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.bed") %>% dplyr::select(V1, V2, V3, V4, V5)
colnames(ICGC.snvs.closest.ori.10kb.df) <- c("seqnames", "start", "end", "REF", "ALT")
ICGC.snvs.closest.ori.10kb.gr <- makeGRangesFromDataFrame(ICGC.snvs.closest.ori.10kb.df, keep.extra.columns = T)
ICGC.snvs.closest.ori.10kb.gr$score <- 1
ICGC.snvs.closest.ori.cluster.50kb.df <- read.table("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.cluster.50kb.bed") %>% dplyr::select(V1, V2, V3, V4, V5)
colnames(ICGC.snvs.closest.ori.cluster.50kb.df) <- c("seqnames", "start", "end", "REF", "ALT")
ICGC.snvs.closest.ori.cluster.50kb.gr <- makeGRangesFromDataFrame(ICGC.snvs.closest.ori.cluster.50kb.df, keep.extra.columns = T)
ICGC.snvs.closest.ori.cluster.50kb.gr$score <- 1
ori.isol.snvs.df <- as.data.frame(mergeByOverlaps(ori.isol.gr, ICGC.snvs.closest.ori.10kb.gr)) %>% group_by(ori.id) %>% summarise(snvs.count = n()) %>% mutate(class = "isolated")
ori.cluster.snvs.df <- as.data.frame(mergeByOverlaps(ori.cluster.gr, ICGC.snvs.closest.ori.cluster.50kb.gr)) %>% group_by(ori.id) %>% summarise(snvs.count = n()) %>% mutate(class = "cluster")
ori.cluster.summary.snvs.df <- rbind(ori.isol.snvs.df, ori.cluster.snvs.df)
saveRDS(ori.cluster.summary.snvs.df, "/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/ori.cluster.summary.snvs.df.rds")
ks.test(ori.isol.snvs.df$snvs.count, ori.cluster.snvs.df$snvs.count, alternative = "less")
# p-value = 0.8949Plot the distribution of mutations at origin clusters and compare mutational burden
library(dplyr)
library(tidyr)
library(ggplot2)
library(ggpubr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load df
snvs.ori.cluster.mat <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/snvs.ori.cluster.mat.rds")
# Plot
snvs.ori.cluster.df <- as.data.frame(snvs.ori.cluster.mat) %>%
summarise_all(~ mean(.x, na.rm = TRUE)) %>% t() %>% `colnames<-`(.[1, ]) %>% .[-1, ] %>% as_tibble() %>% mutate(bin = seq(1,199,1)) %>%
mutate_if(is.character, as.numeric) %>% mutate(across(-bin, ~ ./mean(.[1:40])))
snvs.ori.cluster.plot <- snvs.ori.cluster.df %>% ggplot(aes(x=bin, y=as.numeric(value))) +
geom_line(size = 0.75) +
ylab("Mutation enrichment\nover background (log2 FC)") +
theme_bw() + theme(aspect.ratio=0.5, panel.grid.minor = element_line(linetype = "dotted")) +
scale_x_continuous(name="Origin clusters", limits=c(0, 200), n.breaks = 6, labels = c("-50kb", "start", "50%", "end", "+50kb"))
snvs.ori.cluster.plot
# Laod mutational burden data
ori.cluster.summary.snvs.df <- readRDS( "/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/ori.cluster.summary.snvs.df.rds")
ori.cluster.summary.snvs.plot <- ori.cluster.summary.snvs.df %>% ggplot(aes(x=class, y=snvs.count, fill = class, alpha = 0.3)) +
geom_boxplot(width = 0.5, fill="#D56114") +
scale_y_continuous(trans='log2', limits = c(1,1000)) + ylab("snvs count") + ggtitle("p-value = 0.8949") +
theme_bw() + theme(aspect.ratio=2, panel.grid.minor = element_line(linetype = "dotted"), legend.position = "none", axis.title.x=element_blank(), axis.text.x=element_text(angle=45,hjust=1))
ori.cluster.summary.snvs.plot
pdf("/Users/pm23/Desktop/Projects/OriCan/Manuscript/Nature Communications/Rplots/origin.clusters.pdf", width=8, height=4, useDingbats=FALSE)
ggarrange(snvs.ori.cluster.plot, ori.cluster.summary.snvs.plot, nrow = 1)
dev.off()## quartz_off_screen
## 22.3 Origin mutation rate
Compute snvs mutation rate for GC/AT mutations and each pyrimidine mutation corrected by the base composition at origin
# r
# Load base composition statistics
ori.bc.summary <- read.csv("./Dataset/ori/ori.bc.summary.csv")
# For GC/AT mutations
ICGC.snvs.ori.mut.rate.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
mutate(mut.type = ifelse((V4 == "G" | V4 == "C"), "GC", "AT")) %>% group_by(dist) %>%
summarise(GC.mut = sum(mut.type == "GC"), AT.mut = sum(mut.type == "AT")) %>%
right_join(ori.bc.summary) %>%
mutate(GC.rate = GC.mut/(G+C), AT.rate=AT.mut/(A+T)) %>%
mutate(GC.rate.fold=log2(GC.rate/mean(GC.rate[c(1:20,180:200)])), AT.rate.fold=log2(AT.rate/mean(AT.rate[c(1:20,180:200)])))
ICGC.GC.snvs.ori.mut.rate.df <- ICGC.snvs.ori.mut.rate.df %>% dplyr::select(dist, rate = GC.rate.fold) %>% mutate(type = "GC")
ICGC.AT.snvs.ori.mut.rate.df <- ICGC.snvs.ori.mut.rate.df %>% dplyr::select(dist, rate = AT.rate.fold) %>% mutate(type = "AT")
# For each pyrimidine mutations
# C>A
ICGC.CA.ori.mut.rate.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "C" & V5 == "A")|(V4 == "G" | V5 == "T")) %>%
group_by(dist) %>% summarise(CA.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(CA.rate=CA.mut/(G+C), CA.rate.fold=log2(CA.rate/mean(CA.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CA.rate.fold) %>% mutate(type = "C>A")
# C>G
ICGC.CG.ori.mut.rate.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "C" & V5 == "G")|(V4 == "G" | V5 == "C")) %>%
group_by(dist) %>% summarise(CG.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(CG.rate=CG.mut/(G+C), CG.rate.fold=log2(CG.rate/mean(CG.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CG.rate.fold) %>% mutate(type = "C>G")
# C>T
ICGC.CT.ori.mut.rate.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "C" & V5 == "T")|(V4 == "G" | V5 == "A")) %>%
group_by(dist) %>% summarise(CT.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(CT.rate=CT.mut/(G+C), CT.rate.fold=log2(CT.rate/mean(CT.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CT.rate.fold) %>% mutate(type = "C>T")
# T>A
ICGC.TA.ori.mut.rate.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "T" & V5 == "A")|(V4 == "A" | V5 == "T")) %>%
group_by(dist) %>% summarise(TA.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(TA.rate=TA.mut/(T+A), TA.rate.fold=log2(TA.rate/mean(TA.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TA.rate.fold) %>% mutate(type = "T>A")
# T>C
ICGC.TC.ori.mut.rate.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "T" & V5 == "C")|(V4 == "A" | V5 == "G")) %>%
group_by(dist) %>% summarise(TC.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(TC.rate=TC.mut/(T+A), TC.rate.fold=log2(TC.rate/mean(TC.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TC.rate.fold) %>% mutate(type = "T>C")
# T>G
ICGC.TG.ori.mut.rate.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "T" & V5 == "G")|(V4 == "A" | V5 == "C")) %>%
group_by(dist) %>% summarise(TG.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(TG.rate=TG.mut/(T+A), TG.rate.fold=log2(TG.rate/mean(TG.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TG.rate.fold) %>% mutate(type = "T>G")
# Prepare summary df
ICGC.snvs.ori.mut.summary.rate.df <- rbind(ICGC.GC.snvs.ori.mut.rate.df, ICGC.AT.snvs.ori.mut.rate.df,
ICGC.CA.ori.mut.rate.df, ICGC.CG.ori.mut.rate.df, ICGC.CT.ori.mut.rate.df,
ICGC.TA.ori.mut.rate.df, ICGC.TC.ori.mut.rate.df, ICGC.TG.ori.mut.rate.df)
saveRDS(ICGC.snvs.ori.mut.summary.rate.df, "./001_Mut_density_Pan_cancer/rds/ICGC.snvs.ori.mut.summary.rate.df.rds")Plot
library(dplyr)
library(tidyr)
library(ggplot2)
library(ggpubr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load df
ICGC.snvs.ori.mut.summary.rate.df <- readRDS("./001_Mut_density_Pan_cancer/rds/ICGC.snvs.ori.mut.summary.rate.df.rds")
# Plot GC>AT and AT>GC mutation rates
ICGC.GC.AT.rate.plot <- ICGC.snvs.ori.mut.summary.rate.df %>% filter(type == "GC" | type == "AT") %>%
ggplot(aes(x=dist, y=rate, colour=type)) + geom_line() +
scale_color_manual(values = c("#E41F1A", "#4F4A75")) +
xlim(-5000,5000) +
xlab("Distance from origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.GC.AT.rate.plot
# Plot pyrimidine mutation rates
ICGC.pyr.rate.plot <- ICGC.snvs.ori.mut.summary.rate.df %>% filter(type != "GC" & type != "AT") %>%
ggplot(aes(x=dist, y=rate, colour=type)) + geom_line() +
scale_color_manual(values=c("#E69F00", "#56B4E9", "#009E73","#CC79A7", "#0072B2", "#D55E00")) +
xlim(-5000,5000) +
xlab("Distance from origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.pyr.rate.plot
pdf("./Rplots/ICGC.pyr.rate.plot.pdf", width=7, height=6, useDingbats=FALSE)
ICGC.pyr.rate.plot
dev.off()## quartz_off_screen
## 2Compute mutation rates corrected for trinucleotide composition
# R
# Format mutation coordinates in 100 bp windows at origins
ICGC.snvs.closest.ori.10kb.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.bed")
ICGC.snvs.closest.ori.reform.10kb.df <- ICGC.snvs.closest.ori.10kb.df %>%
dplyr::select(seqnames = V1, start = V2, end = V3, REF = V4, ALT = V5, dist = V14)
# Define windows
ICGC.snvs.closest.ori.windows.10kb.df <- ICGC.snvs.closest.ori.reform.10kb.df %>% mutate(dist = -dist) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100)
# Recover sequence context
ICGC.snvs.closest.ori.windows.10kb.gr <- makeGRangesFromDataFrame(ICGC.snvs.closest.ori.windows.10kb.df, keep.extra.columns = T)
seqlevelsStyle(ICGC.snvs.closest.ori.windows.10kb.gr) <- "UCSC"
ICGC.snvs.closest.ori.windows.resize.10kb.gr <- resize(ICGC.snvs.closest.ori.windows.10kb.gr, 1, fix = "start")+1
snvs.seq.context <- getSeq(Hsapiens, ICGC.snvs.closest.ori.windows.resize.10kb.gr)
snvs.seq.context.df <- tibble(dist = ICGC.snvs.closest.ori.windows.resize.10kb.gr$dist,
REF = ICGC.snvs.closest.ori.windows.resize.10kb.gr$REF,
ALT = ICGC.snvs.closest.ori.windows.resize.10kb.gr$ALT,
context = as.character(snvs.seq.context))
snvs.seq.context.split.df <- snvs.seq.context.df %>%
separate(context, c("UP", "REF.context", "DOWN"), sep = "(?<=.)", extra = "drop") %>%
mutate(trinuc = paste0(UP, REF.context, DOWN))
snvs.seq.context.split.2.df <- snvs.seq.context.split.df %>%
dplyr::select(dist, UP, REF, ALT, DOWN, trinuc)
saveRDS(snvs.seq.context.split.2.df, "./001_Mut_density_Pan_cancer/rds/snvs.seq.context.split.df.rds")
# Compute trinucleotide base composition at origins
ori.10kb.domain.100nt.split.gr <- import("./Dataset/ori/ori.10kb.domain.100nt.split.hg19.NCBI.bed")
my.chr <- c(1:22, "X", "Y")
ori.10kb.domain.100nt.split.gr <- ori.10kb.domain.100nt.split.gr[seqnames(ori.10kb.domain.100nt.split.gr) %in% my.chr]
seqlevelsStyle(ori.10kb.domain.100nt.split.gr) <- "UCSC"
ori.views <- Views(Hsapiens, ori.10kb.domain.100nt.split.gr)
ori.trinuc <- trinucleotideFrequency(ori.views, step=1, as.prob=FALSE, as.array=FALSE, fast.moving.side="right")
ori.trinuc.df <- cbind.data.frame(bin = ori.10kb.domain.100nt.split.gr$name, ori.trinuc)
ori.trinuc.summary.df <- ori.trinuc.df %>% group_by(bin) %>% summarise(across(everything(), sum)) %>% mutate(dist = (as.numeric(bin)-100)*100)
saveRDS(ori.trinuc.summary.df, "./001_Mut_density_Pan_cancer/rds/ori.trinuc.summary.df.rds")
# Melt df
ori.trinuc.summary.df <- readRDS("./001_Mut_density_Pan_cancer/rds/ori.trinuc.summary.df.rds")
ori.trinuc.summary.melt.df <- ori.trinuc.summary.df %>% dplyr::select(-bin) %>%
gather("trinuc", "count.trinuc", -dist)
# Combine information
snvs.seq.context.split.2.df <- readRDS("./001_Mut_density_Pan_cancer/rds/snvs.seq.context.split.df.rds")
snvs.seq.context.summary.df <- snvs.seq.context.split.2.df %>%
filter(ALT != ".") %>%
mutate(mut.type = paste(REF, ALT, sep = ">")) %>%
mutate(mut.type = case_when(mut.type == "G>A" ~ "C>T",
mut.type == "G>C" ~ "C>G",
mut.type == "G>T" ~ "C>A",
mut.type == "A>C" ~ "T>G",
mut.type == "A>G" ~ "T>C",
mut.type == "A>T" ~ "T>A",
T ~ mut.type))
# Compute corrected frequency for each mutation type
snvs.trinuc.freq.df <- snvs.seq.context.summary.df %>% ungroup() %>% group_by(mut.type, trinuc, dist) %>% summarise(count = n()) %>%
left_join(ori.trinuc.summary.melt.df, by = c("dist", "trinuc")) %>% ungroup() %>%
group_by(dist, mut.type) %>% mutate(trinuc.corr = count/count.trinuc) %>%
summarise(rate = sum(trinuc.corr))
# Correct for background values
snvs.trinuc.corr.freq.df <- snvs.trinuc.freq.df %>% ungroup() %>% group_by(mut.type) %>%
mutate(rate.fold=log2(rate/mean(rate[c(1:20,180:200)])))
saveRDS(snvs.trinuc.corr.freq.df, "./001_Mut_density_Pan_cancer/rds/snvs.trinuc.corr.freq.df.rds")
# Focus on C>T mutation to distinguish CpG and non-CpG context
CtoT.trinuc.freq.df <- snvs.seq.context.summary.df %>% ungroup() %>% filter(mut.type == "C>T") %>%
mutate(context = case_when(REF == "C" & DOWN == "G" ~ "CpG",
REF == "G" & UP == "C" ~ "CpG",
T ~ "non CpG")) %>%
group_by(trinuc, dist, context) %>% summarise(count = n()) %>%
left_join(ori.trinuc.summary.melt.df, by = c("dist", "trinuc")) %>% ungroup() %>%
group_by(dist, context) %>% mutate(trinuc.corr = count/count.trinuc) %>%
summarise(rate = sum(trinuc.corr)) %>% ungroup() %>% group_by(context) %>%
mutate(rate.fold=log2(rate/mean(rate[c(1:20,180:200)])))
saveRDS(CtoT.trinuc.freq.df, "./001_Mut_density_Pan_cancer/rds/CtoT.trinuc.freq.df.rds")Plot
library(dplyr)
library(tidyr)
library(ggplot2)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load results
snvs.trinuc.corr.freq.df <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/snvs.trinuc.corr.freq.df.rds")
CtoT.trinuc.freq.df <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/CtoT.trinuc.freq.df.rds")
# Plot
snvs.trinuc.corr.freq.plot <- snvs.trinuc.corr.freq.df %>%
ggplot(aes(x=dist, y=rate.fold, colour=mut.type)) + geom_line() +
scale_color_manual(values=c("#E69F00", "#56B4E9", "#009E73","#CC79A7", "#0072B2", "#D55E00")) +
xlim(-5000,5000) +
xlab("Distance from origin (bp)") + ylab("Trinucleotide frequency corrected\nmutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins corrected for trinuc frequency") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
snvs.trinuc.corr.freq.plot
CtoT.trinuc.freq.plot <- CtoT.trinuc.freq.df %>%
ggplot(aes(x=dist, y=rate.fold, colour=context)) + geom_line() +
scale_color_manual(values=c("#EF4136", "#3E54A4")) +
xlim(-5000,5000) + ylim(-1.5, 0.5) +
xlab("Distance from origin (bp)") + ylab("Trinucleotide frequency corrected\nC>T mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins corrected for trinuc frequency") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
CtoT.trinuc.freq.plot
# Save plot
pdf("/Users/pm23/Desktop/Projects/OriCan/Manuscript/Nature Communications/Rplots/ICGC.mut.rate.trinuc.plot.pdf", width=12, height=6, useDingbats=FALSE)
ggarrange(snvs.trinuc.corr.freq.plot, CtoT.trinuc.freq.plot, nrow = 1)
dev.off()## quartz_off_screen
## 22.4 Allele frequency at origins
Assess the distribution of snvs allele frequency at origins
# r
# Load snvs data
ICGC.snvs.closest.ori.10kb.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.bed")
ICGC.snvs.closest.ori.10kb.reshuffle.df <- read.table("./001_Mut_density_Pan_cancer/ICGC.snvs.closest.ori.10kb.reshuffle.bed")
# Compute mean minor allele frequency (MAF) in bin of 100 nt
# MAF is computed as the number of occurences of a given mutations divided by the total of screened patients (19729)
ICGC.snvs.AF.ori.df <- ICGC.snvs.closest.ori.10kb.df %>% mutate(dist = -V14, MAF = (V7*V8)/19729) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
group_by(dist) %>% summarise(MAF = mean(MAF, na.rm = T)) %>% mutate(class = "ori")
ICGC.snvs.AF.ori.reshuffle.df <- ICGC.snvs.closest.ori.10kb.reshuffle.df %>% mutate(dist = -V14, MAF = (V7*V8)/19729) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
group_by(dist) %>% summarise(MAF = mean(MAF, na.rm = T)) %>% mutate(class = "ori.reshuffle")
# Combine
ICGC.snvs.AF.ori.summary.df <- rbind(ICGC.snvs.AF.ori.df, ICGC.snvs.AF.ori.reshuffle.df)
saveRDS(ICGC.snvs.AF.ori.summary.df, "./001_Mut_density_Pan_cancer/rds/ICGC.snvs.AF.ori.summary.df.rds")Plot
library(dplyr)
library(tidyr)
library(ggplot2)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load df
ICGC.snvs.AF.ori.summary.df <- readRDS("./001_Mut_density_Pan_cancer/rds/ICGC.snvs.AF.ori.summary.df.rds")
# Plot
ICGC.snvs.AF.ori.summary.plot <- ICGC.snvs.AF.ori.summary.df %>% ggplot(aes(x=dist, y=MAF, colour=class)) + geom_line() +
scale_color_manual(values = c("#E41F1A", "#4F4A75")) +
xlim(-5000,5000) +
xlab("Distance from origin (bp)") + ylab("Mean minor allele frequency") + ggtitle("snvs allele frequency at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.snvs.AF.ori.summary.plot
pdf("./Rplots/ICGC.snvs.AF.ori.summary.plot.pdf", width=7, height=6, useDingbats=FALSE)
ICGC.snvs.AF.ori.summary.plot
dev.off()## quartz_off_screen
## 22.5 At SNS core origins
Assess mutagenesis at SNS-seq origins
# R
# Format SNS-seq core origins
SNS.core.hg38.df <- read.table("./Dataset/ori/GSE128477_Core_origins_hg38.bed")
colnames(SNS.core.hg38.df) <- c("seqnames", "start", "end", "ori.id")
SNS.core.hg38.gr <- makeGRangesFromDataFrame(SNS.core.hg38.df, keep.extra.columns = T)
# Liftover to hg19
hg38ToHg19.chain <- import.chain("./Dataset/ori/hg38ToHg19.over.chain")
SNS.core.hg19.gr <- unlist(liftOver(SNS.core.hg38.gr, hg38ToHg19.chain)) # 65,329 origins
# Filter out SNS core origins overlapping with constitutive ones
ori.center.df <- read.table("./Dataset/ori/ori.center.hg19.NCBI.sorted.bed")
colnames(ori.center.df) <- c("seqnames", "start", "end", "EFF", "ori.id", "ori.width")
ori.df <- ori.center.df %>% mutate(start = round(start-(ori.width/2)), end = round(end+(ori.width/2)))
ori.gr <- makeGRangesFromDataFrame(ori.df)
seqlevelsStyle(ori.gr) <- "UCSC"
SNS.core.overlap <- findOverlaps(SNS.core.hg19.gr, ori.gr, 5000L)
SNS.core.filter.hg19.gr <- SNS.core.hg19.gr[-queryHits(SNS.core.overlap)] # 32,425 origins
seqlevelsStyle(SNS.core.filter.hg19.gr) <- "NCBI"
# Save bed file
SNS.core.filter.hg19.bed <- as.data.frame(SNS.core.filter.hg19.gr) %>% select(seqnames, start, end)
write.table(SNS.core.filter.hg19.bed, "./Dataset/ori/SNS.core.hg19.NCBI.bed", sep="\t", col.names = F, row.names = F, quote = F)
# Compute SNS core origin midpoints
SNS.core.midpoints.hg19.bed <- SNS.core.filter.hg19.bed %>%
mutate(start = as.integer(round((start+end)/2)), end = as.integer(start+1))
write.table(SNS.core.midpoints.hg19.bed, "./Dataset/ori/SNS.core.midpoint.hg19.NCBI.bed", sep="\t", col.names = F, row.names = F, quote = F)
# SNS-core origin filtering
SNS.core.center.df <- read.table("./Dataset/ori/SNS.core.midpoint.hg19.NCBI.bed")
SNS.core.inter.dist.df <- SNS.core.center.df %>% mutate(prev = V2-lag(V2), nxt = lead(V2)-V2) %>%
mutate(min = pmin(prev, nxt, na.rm = T)) %>% mutate(min = ifelse(min > 0, min, NA)) %>%
filter(min >= 20000) %>% dplyr::select(-prev, -nxt, -min)
# 12,041 remaining origins
write.table(SNS.core.inter.dist.df, "./Dataset/ori/SNS.core.inter.dist.bed", sep="\t", col.names = F, row.names = F, quote = F)
# Base composition statistics
SNS.core.10kb.df <- SNS.core.inter.dist.df %>% mutate(V2 = as.integer(V2-10000), V3 = as.integer(V3+9999))
write.table(SNS.core.10kb.df, "./Dataset/ori/SNS.core.10kb.domain.hg19.NCBI.bed", sep="\t", col.names = F, row.names = F, quote = F)
/Users/pm23/Desktop/Software/bedtools2/bin/bedtools makewindows -b /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/SNS.core.10kb.domain.hg19.NCBI.bed \
-w 100 -i winnum > /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/SNS.core.10kb.domain.100nt.split.hg19.NCBI.bed
SNS.core.10kb.domain.100nt.split.gr <- import("./Dataset/ori/SNS.core.10kb.domain.100nt.split.hg19.NCBI.bed")
my.chr <- c(1:22, "X", "Y")
SNS.core.10kb.domain.100nt.split.gr <- SNS.core.10kb.domain.100nt.split.gr[seqnames(SNS.core.10kb.domain.100nt.split.gr) %in% my.chr]
seqlevelsStyle(SNS.core.10kb.domain.100nt.split.gr) <- "UCSC"
# Recover sequences and compute base composition statistics
SNS.core.views <- Views(Hsapiens, SNS.core.10kb.domain.100nt.split.gr)
SNS.core.bc <- letterFrequency(SNS.core.views, c("A", "C", "G", "T"), as.prob = FALSE)
SNS.core.bc.df <- cbind.data.frame(bin = SNS.core.10kb.domain.100nt.split.gr$name, SNS.core.bc)
SNS.core.bc.summary <- SNS.core.bc.df %>% group_by(bin) %>% summarise_at(c("A", "C", "G", "T"), sum) %>%
mutate(dist = (as.numeric(bin)-100)*100,
A.freq = A/(A+C+T+G),
C.freq = C/(A+C+T+G),
G.freq = G/(A+C+T+G),
T.freq = T/(A+C+T+G),
GC=(G+C)/(A+C+T+G))
write.csv(SNS.core.bc.summary, "./Dataset/ori/SNS.core.bc.summary.csv", quote = F, row.names = F)Compute snvs distances to SNS core origins
# bash
sort -k1,1 -k2,2n /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/SNS.core.inter.dist.bed > /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/SNS.core.inter.dist.sorted.bed
/Users/pm23/Desktop/Software/bedtools2/bin/bedtools closest \
-a /Users/pm23/Desktop/Projects/OriCan/Dataset/ICGC/SSM/ICGC.snvs.aggregated.sorted.bed \
-b /Users/pm23/Desktop/Projects/OriCan/Dataset/ori/SNS.core.inter.dist.sorted.bed -D ref \
> /Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.SNS.core.bed
# Select mutations in proximity to origins (+- 10kb)
awk '$12>=-10000 && $12<=10000' /Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.SNS.core.bed \
> /Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/ICGC.snvs.closest.SNS.core.10kb.bedCompute mutation rates
# R
# Compute mutation rates
# For GC/AT mutations
ICGC.snvs.SNS.core.mut.rate.df <- ICGC.snvs.closest.SNS.core.10kb.df %>% mutate(dist = -V12) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
mutate(mut.type = ifelse((V4 == "G" | V4 == "C"), "GC", "AT")) %>% group_by(dist) %>%
summarise(GC.mut = sum(mut.type == "GC"), AT.mut = sum(mut.type == "AT")) %>%
right_join(SNS.core.bc.summary) %>%
mutate(GC.rate = GC.mut/(G+C), AT.rate=AT.mut/(A+T)) %>%
mutate(GC.rate.fold=log2(GC.rate/mean(GC.rate[c(1:20,180:200)])), AT.rate.fold=log2(AT.rate/mean(AT.rate[c(1:20,180:200)])))
ICGC.GC.snvs.SNS.core.mut.rate.df <- ICGC.snvs.SNS.core.mut.rate.df %>% dplyr::select(dist, rate = GC.rate.fold) %>% mutate(type = "GC")
ICGC.AT.snvs.SNS.core.mut.rate.df <- ICGC.snvs.SNS.core.mut.rate.df %>% dplyr::select(dist, rate = AT.rate.fold) %>% mutate(type = "AT")
# For each pyrimidine mutations
# C>A
ICGC.CA.SNS.core.mut.rate.df <- ICGC.snvs.closest.SNS.core.10kb.df %>% mutate(dist = -V12) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "C" & V5 == "A")|(V4 == "G" | V5 == "T")) %>%
group_by(dist) %>% summarise(CA.mut=n()) %>% right_join(SNS.core.bc.summary) %>% mutate(CA.rate=CA.mut/(G+C), CA.rate.fold=log2(CA.rate/mean(CA.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CA.rate.fold) %>% mutate(type = "C>A")
# C>G
ICGC.CG.SNS.core.mut.rate.df <- ICGC.snvs.closest.SNS.core.10kb.df %>% mutate(dist = -V12) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "C" & V5 == "G")|(V4 == "G" | V5 == "C")) %>%
group_by(dist) %>% summarise(CG.mut=n()) %>% right_join(SNS.core.bc.summary) %>% mutate(CG.rate=CG.mut/(G+C), CG.rate.fold=log2(CG.rate/mean(CG.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CG.rate.fold) %>% mutate(type = "C>G")
# C>T
ICGC.CT.SNS.core.mut.rate.df <- ICGC.snvs.closest.SNS.core.10kb.df %>% mutate(dist = -V12) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "C" & V5 == "T")|(V4 == "G" | V5 == "A")) %>%
group_by(dist) %>% summarise(CT.mut=n()) %>% right_join(SNS.core.bc.summary) %>% mutate(CT.rate=CT.mut/(G+C), CT.rate.fold=log2(CT.rate/mean(CT.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CT.rate.fold) %>% mutate(type = "C>T")
# T>A
ICGC.TA.SNS.core.mut.rate.df <- ICGC.snvs.closest.SNS.core.10kb.df %>% mutate(dist = -V12) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "T" & V5 == "A")|(V4 == "A" | V5 == "T")) %>%
group_by(dist) %>% summarise(TA.mut=n()) %>% right_join(SNS.core.bc.summary) %>% mutate(TA.rate=TA.mut/(T+A), TA.rate.fold=log2(TA.rate/mean(TA.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TA.rate.fold) %>% mutate(type = "T>A")
# T>C
ICGC.TC.SNS.core.mut.rate.df <- ICGC.snvs.closest.SNS.core.10kb.df %>% mutate(dist = -V12) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "T" & V5 == "C")|(V4 == "A" | V5 == "G")) %>%
group_by(dist) %>% summarise(TC.mut=n()) %>% right_join(SNS.core.bc.summary) %>% mutate(TC.rate=TC.mut/(T+A), TC.rate.fold=log2(TC.rate/mean(TC.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TC.rate.fold) %>% mutate(type = "T>C")
# T>G
ICGC.TG.SNS.core.mut.rate.df <- ICGC.snvs.closest.SNS.core.10kb.df %>% mutate(dist = -V12) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((V4 == "T" & V5 == "G")|(V4 == "A" | V5 == "C")) %>%
group_by(dist) %>% summarise(TG.mut=n()) %>% right_join(SNS.core.bc.summary) %>% mutate(TG.rate=TG.mut/(T+A), TG.rate.fold=log2(TG.rate/mean(TG.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TG.rate.fold) %>% mutate(type = "T>G")
# Prepare summary df
ICGC.snvs.SNS.core.mut.summary.rate.df <- rbind(ICGC.GC.snvs.SNS.core.mut.rate.df, ICGC.AT.snvs.SNS.core.mut.rate.df,
ICGC.CA.SNS.core.mut.rate.df, ICGC.CG.SNS.core.mut.rate.df, ICGC.CT.SNS.core.mut.rate.df,
ICGC.TA.SNS.core.mut.rate.df, ICGC.TC.SNS.core.mut.rate.df, ICGC.TG.SNS.core.mut.rate.df)
saveRDS(ICGC.snvs.SNS.core.mut.summary.rate.df, "./001_Mut_density_Pan_cancer/rds/ICGC.snvs.SNS.core.mut.summary.rate.df.rds")Plot
library(dplyr)
library(tidyr)
library(ggplot2)
# Load df
ICGC.snvs.SNS.core.mut.summary.rate.df <- readRDS( "/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/ICGC.snvs.SNS.core.mut.summary.rate.df.rds")
# Plot GC>AT and AT>GC mutation rates
ICGC.GC.AT.rate.SNS.core.plot <- ICGC.snvs.SNS.core.mut.summary.rate.df %>% filter(type == "GC" | type == "AT") %>%
ggplot(aes(x=dist, y=rate, colour=type)) + geom_line() +
scale_color_manual(values = c("#E41F1A", "#4F4A75")) +
xlim(-5000,5000) +
xlab("Distance from SNS-core origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at SNS core origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.GC.AT.rate.SNS.core.plot
# Plot pyrimidine mutation rates (same scale as figure 1a to allow comparison)
ICGC.pyr.rate.SNS.core.plot <- ICGC.snvs.SNS.core.mut.summary.rate.df %>% filter(type != "GC" & type != "AT") %>%
ggplot(aes(x=dist, y=rate, colour=type)) + geom_line() +
scale_color_manual(values=c("#E69F00", "#56B4E9", "#009E73","#CC79A7", "#0072B2", "#D55E00")) +
xlim(-5000,5000) + ylim(-0.15,1.65) +
xlab("Distance from SNS-core origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at SNS-core origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.pyr.rate.SNS.core.plot
# Save plot
pdf("/Users/pm23/Desktop/Projects/OriCan/Manuscript/Nature Communications/Rplots/ICGC.pyr.rate.SNS.core.plot.pdf", width=7, height=6, useDingbats=FALSE)
ICGC.pyr.rate.SNS.core.plot
dev.off()## quartz_off_screen
## 23 Pan-cancer analysis of mutation rates at origins
3.1 Mutation density at origins versus flanks by primary sites
Prepare bed files.
# r
# Load bed files
ori.10kb.domains.df <- read.table("./Dataset/ori/ori.10kb.domain.hg19.NCBI.bed", sep="\t")
colnames(ori.10kb.domains.df) <- c("seqnames", "start", "end", "EFF", "ori.id")
ori.1kb.domains.df <- read.table("./Dataset/ori/ori.1kb.domain.hg19.NCBI.bed" , sep="\t")
colnames(ori.1kb.domains.df) <- c("seqnames", "start", "end", "EFF", "ori.id")
# Create gr
my.chr <- c(1:22, "X", "Y")
ori.10kb.domains.gr <- makeGRangesFromDataFrame(ori.10kb.domains.df, keep.extra.columns = T)
ori.10kb.domains.gr <- ori.10kb.domains.gr[seqnames(ori.10kb.domains.gr) %in% my.chr]
ori.1kb.domains.gr <- makeGRangesFromDataFrame(ori.1kb.domains.df, keep.extra.columns = T)
ori.1kb.domains.gr <- ori.1kb.domains.gr[seqnames(ori.1kb.domains.gr) %in% my.chr]
# Compute flank coordinates
ori.flanks.gr <- setdiff(ori.10kb.domains.gr, ori.1kb.domains.gr, ignore.strand=TRUE)
# Save gr
saveRDS(ori.1kb.domains.gr, "./001_Mut_density_Pan_cancer/rds/ori.1kb.domains.gr.rds")
saveRDS(ori.flanks.gr, "./001_Mut_density_Pan_cancer/rds/ori.flanks.gr.rds")
# Prepare bed files
ori.1kb.domains.bed <- as.data.frame(ori.1kb.domains.gr) %>% dplyr::select(seqnames, start, end)
ori.flanks.bed <- as.data.frame(ori.flanks.gr) %>% dplyr::select(seqnames, start, end)
# Save bed files
write.table(ori.1kb.domains.bed, "./Dataset/ori/BED/ori.1kb.hg19.bed", sep="\t", col.names = F, row.names = F, quote = F)
write.table(ori.flanks.bed, "./Dataset/ori/BED/ori.flanks.hg19.bed", sep="\t", col.names = F, row.names = F, quote = F)Recover individual vcf files
Generated manifest from the ICGC data portal using the following filtering criteria: SSM > Collaboratory - Toronto > WGS > VCF > Broad variant call pipeline –> 3,900 files from 1,830 donors (/cephfs2/pmurat/OriCan/Dataset/ICGC/SSM/manifest.1698683356752.tar.gz)
Load and modify the manifest
# r
library(dplyr)
library(stringr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
#
SSM.manifest.df <- read.table(gzfile("./Dataset/ICGC/SSM/manifest.1698683356752.tar.gz"), skip = 1)
SSM.manifest.df <- SSM.manifest.df %>% dplyr::select(V9, V10, V2, V3, V5)
colnames(SSM.manifest.df) <- c("donor.id", "project", "file.id", "object.id", "file.name")
# Select snvs
snvs.manifest.df <- SSM.manifest.df %>% filter(str_detect(file.name, 'somatic.snv')) %>% separate(project, c("cancer.type", "project"), sep = "-") # 1,950 entries, 1,830 donors
# Save manifest
write.table(snvs.manifest.df, "./Dataset/ICGC/SSM/snvs.manifest.tsv", sep="\t", col.names = F, row.names = F, quote = F)Download files with score-client using the /cephfs2/pmurat/OriCan/Scripts/snvs.score.client.sh
Process data snvs count are reported as the number of snvs per kb
# r
library(tidyr)
# Open ori and flanks coordinates
ori.1kb.domains.gr <- import("./Dataset/ori/BED/ori.1kb.hg19.bed")
ori.flanks.gr <- import("./Dataset/ori/BED/ori.flanks.hg19.bed")
# Compute gr width
sum(width(ori.1kb.domains.gr)) # 9,340,000 bp
sum(width(ori.flanks.gr)) # 177,441,320 bp
# List vcf files
vcf.files <- list.files("./Dataset/ICGC/SSM/VCF/") # 3,892 files
vcf.files <- vcf.files[!grepl(".idx", vcf.files)] # 3,892 snvs vcf files
# Compute snvs density ratios
snvs.dens.ratio.df <- tibble()
for (i in 1:length(vcf.files)) {
vcf.files.i <- vcf.files[i]
print(vcf.files.i)
df.i <- snvs.manifest.df %>% filter(file.name == vcf.files.i)
donor.i <- df.i$donor.id
cancer.type.i <- df.i$cancer.type
project.i <- df.i$project
path.i <- paste("./Dataset/ICGC/SSM/VCF/", vcf.files.i, sep = "")
vcf.i <- read.table(gzfile(path.i), comment.char = "#") %>% dplyr::select(V1, V2, V4, V5)
count.i <- nrow(vcf.i)
bed.i <- vcf.i %>% mutate(end = V2 + 1) %>% dplyr::select(seqnames = V1, start = V2, end, REF = V4, ALT = V5)
gr.i <- makeGRangesFromDataFrame(bed.i)
ori.overlap.i <- subsetByOverlaps(gr.i, ori.1kb.domains.gr)
snvs.ori.count.i <- (length(ori.overlap.i)*1000)/9340000
flanks.overlap.i <- subsetByOverlaps(gr.i, ori.flanks.gr)
snvs.flanks.count.i <- (length(flanks.overlap.i)*1000)/177441320
df.summary.i <- cbind.data.frame(donor = donor.i, cancer.type = cancer.type.i, cancer.project = project.i, snvs.count = count.i, snvs.dens.ori = snvs.ori.count.i, snvs.dens.flanks = snvs.flanks.count.i) %>% mutate(snvs.dens.ratio = snvs.dens.ori/snvs.dens.flanks)
snvs.dens.ratio.df <- rbind(snvs.dens.ratio.df,df.summary.i)
}
saveRDS(snvs.dens.ratio.df, "./001_Mut_density_Pan_cancer/rds/snvs.dens.ratio.df.rds")Analyse results - genomes with less than 5,000 snvs are filtered out. We retained 1,056 genomes.
library(dplyr)
library(tidyr)
library(ggplot2)
library(scales)
library(plotrix)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load snvs density ratio df
snvs.dens.ratio.df <- readRDS("./001_Mut_density_Pan_cancer/rds/snvs.dens.ratio.df.rds")
# DONE PREVIOUSLY
# # Load cancer project information
# cancer.project.info.df <- read.csv("./Dataset/ICGC/projects_2023_10_30_04_53_06.tsv", sep = "\t") %>% separate(Project.Code, c("cancer.type", "project"), sep = "-") %>% dplyr::select(cancer.type, Primary.Site)
# # Add primary site information
# snvs.dens.ratio.df <- snvs.dens.ratio.df %>% left_join(cancer.project.info.df, by = "cancer.type")
# Compute stats and means
snvs.dens.ratio.summary.df <- snvs.dens.ratio.df %>% group_by(Primary.Site) %>% summarise(mean=mean(snvs.dens.ratio, na.rm=T), min=min(snvs.dens.ratio,na.rm=T), max=max(snvs.dens.ratio,na.rm=T)) %>% arrange(-mean)
# Prepare plot
# Stratified by primary sites
snvs.dens.ratio.plot <- snvs.dens.ratio.df %>% filter(snvs.count >= 5000) %>% distinct() %>%
ggplot(aes(x=reorder(Primary.Site,-snvs.dens.ratio, na.rm=T), y=snvs.dens.ratio, fill="#E41F1A", alpha = 0.3)) +
geom_boxplot(outlier.shape = NA, width = 0.5) +
geom_jitter(color="black", size=0.4, alpha=0.4) + ylab("Mutation density ratio (ori/flanks)") +
geom_hline(yintercept=1, linetype="dashed") +
theme_bw() + theme(aspect.ratio=0.4, panel.grid.minor = element_line(linetype = "dotted"), legend.position = "none", axis.title.x=element_blank(), axis.text.x=element_text(angle=45,hjust=1))
snvs.dens.ratio.plot
# Stratified by cancer types
snvs.dens.ratio.plot.2 <- snvs.dens.ratio.df %>% mutate(cancer.info = paste(cancer.type, Primary.Site, sep = " | ")) %>% filter(snvs.count >= 5000) %>% distinct() %>% ggplot(aes(x=reorder(cancer.info,-snvs.dens.ratio, na.rm=T), y=snvs.dens.ratio, fill="#E41F1A", alpha = 0.3)) +
geom_boxplot(outlier.shape = NA, width = 0.5) +
geom_jitter(color="black", size=0.4, alpha=0.4) + ylab("Mutation density ratio (ori/flanks)") +
geom_hline(yintercept=1, linetype="dashed") +
theme_bw() + theme(aspect.ratio=0.4, panel.grid.minor = element_line(linetype = "dotted"), legend.position = "none", axis.title.x=element_blank(), axis.text.x=element_text(angle=45,hjust=1))
snvs.dens.ratio.plot.2
pdf("./Rplots/snvs.dens.ratio.plot.pdf", width=10, height=6, useDingbats=FALSE)
snvs.dens.ratio.plot.2
dev.off()## quartz_off_screen
## 2# Assess the corelation between the total number of mapped snvs and the snvs ratio at origins
snvs.dens.ratio.plot.3 <- snvs.dens.ratio.df %>% filter(snvs.dens.ratio > 0) %>% distinct() %>%
ggplot(aes(x=snvs.count, y=snvs.dens.ratio)) +
geom_point(alpha = 0.2) + scale_x_continuous(trans = log10_trans(),
breaks = trans_breaks("log10", function(x) 10^x),
labels = trans_format("log10", math_format(10^.x))) + geom_smooth(span = 1e-6, se = T) +
geom_hline(yintercept=1, linetype="dashed") + ylab("Mutation density ratio (ori/flanks)") + xlab("Total snvs count") +
theme_bw() + theme(aspect.ratio=1, panel.grid.minor = element_line(linetype = "dotted"))
snvs.dens.ratio.plot.3
# Same analysis binning the total number of mutations
snvs.dens.ratio.plot.summary.df <- snvs.dens.ratio.df %>% mutate(snvs.total.bin = ntile(snvs.count, 15)) %>% group_by(snvs.total.bin) %>%
summarise(snvs.total.count = mean(snvs.count, na.rm = T), snvs.dens.ratio.mean = mean(snvs.dens.ratio, na.rm = T), snvs.dens.ratio.sd = std.error(snvs.dens.ratio, na.rm = T))
#
snvs.dens.ratio.plot.4 <- snvs.dens.ratio.plot.summary.df %>% ggplot(aes(x=snvs.total.count, y=snvs.dens.ratio.mean)) +
geom_line(color = "#4F4A75") +
geom_point(shape = 21, size = 2, color = "#4F4A75", fill = "white") +
scale_x_continuous(trans = log10_trans(), breaks = trans_breaks("log10", function(x) 10^x), labels = trans_format("log10", math_format(10^.x))) +
geom_errorbar(aes(ymin=snvs.dens.ratio.mean-snvs.dens.ratio.sd, ymax=snvs.dens.ratio.mean+snvs.dens.ratio.sd), width=.1, position=position_dodge(0.05), color = "#4F4A75") +
ylab("Mutation density ratio (ori/flanks)") + xlab("Total snvs count") + ggtitle("Rho = -0.246, p-value < 2.2e-16") +
theme_bw() + theme(aspect.ratio=1, panel.grid.minor = element_line(linetype = "dotted"))
cor.test(log10(snvs.dens.ratio.df$snvs.count), snvs.dens.ratio.df$snvs.dens.ratio) # Rho = -0.2457729, p-value < 2.2e-16##
## Pearson's product-moment correlation
##
## data: log10(snvs.dens.ratio.df$snvs.count) and snvs.dens.ratio.df$snvs.dens.ratio
## t = -15.859, df = 4374, p-value < 2.2e-16
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
## -0.2610027 -0.2049621
## sample estimates:
## cor
## -0.233176snvs.dens.ratio.plot.4
pdf("./Rplots/snvs.dens.ratio.cor.pdf", width=10, height=6, useDingbats=FALSE)
snvs.dens.ratio.plot.4
dev.off()## quartz_off_screen
## 2# Plot also the number of snvs at origins versus the total number of mutations for comparison
snvs.dens.ratio.plot.summary.2.df <- snvs.dens.ratio.df %>% mutate(snvs.total.bin = ntile(snvs.count, 15)) %>% group_by(snvs.total.bin) %>%
summarise(snvs.total.count = mean(snvs.count, na.rm = T), snvs.ori.count.mean = mean(((snvs.dens.ori*9340000)/1000), na.rm = T), snvs.ori.count.sd = std.error(((snvs.dens.ori*9340000)/1000), na.rm = T))
#
snvs.dens.ratio.plot.5 <- snvs.dens.ratio.plot.summary.2.df %>% ggplot(aes(x=snvs.total.count, y=snvs.ori.count.mean)) +
geom_line(color = "#4F4A75") +
geom_point(shape = 21, size = 2, color = "#4F4A75", fill = "white") +
scale_x_continuous(trans = log10_trans(), breaks = trans_breaks("log10", function(x) 10^x), labels = trans_format("log10", math_format(10^.x))) +
scale_y_continuous(trans = log2_trans(), breaks = trans_breaks("log2", function(x) 2^x)) +
geom_errorbar(aes(ymin=snvs.ori.count.mean-snvs.ori.count.sd, ymax=snvs.ori.count.mean+snvs.ori.count.sd), width=.1, position=position_dodge(0.05), color = "#4F4A75") +
ylab("snvs count at origins") + xlab("Total snvs count") + ggtitle("Rho = 0.954, p-value < 2.2e-16") +
theme_bw() + theme(aspect.ratio=1, panel.grid.minor = element_line(linetype = "dotted"))
cor.test(snvs.dens.ratio.df$snvs.count, snvs.dens.ratio.df$snvs.dens.ori) # Rho = 0.9539956, p-value < 2.2e-16##
## Pearson's product-moment correlation
##
## data: snvs.dens.ratio.df$snvs.count and snvs.dens.ratio.df$snvs.dens.ori
## t = 155.71, df = 4374, p-value < 2.2e-16
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
## 0.9157619 0.9248255
## sample estimates:
## cor
## 0.9204173snvs.dens.ratio.plot.5
pdf("./Rplots/snvs.dens.ratio.cor.2.pdf", width=10, height=6, useDingbats=FALSE)
snvs.dens.ratio.plot.5
dev.off()## quartz_off_screen
## 23.2 Mutation rates at origins
Perform mutational burden analysis, corrected for base composition, at origins stratified by cancer types.
We consider only cancer types figuring on previous analysis
Corrected mutation rates are computed by adding the GC>AT and AT>GC mutation rates.
# r
library(tidyr)
# Define cancer types
cancer.projects <- tibble(cancer.projects = unique(ICGC.snvs.closest.ori.10kb.df$V6))
# Select projects
filter <- (snvs.dens.ratio.df %>% filter(snvs.count >= 5000))$cancer.type # 1,056 donors
# Prepare df
cancer.types.df <- cancer.projects %>% separate(cancer.projects, c("cancer.type", "project"), sep = "-") %>% mutate(V6 = paste(cancer.type, project, sep = "-")) %>% filter(cancer.type %in% filter)
# Add information to origin associated snsvs
ICGC.snvs.closest.ori.type.df <- ICGC.snvs.closest.ori.10kb.df %>% left_join(cancer.types.df, by = "V6") %>% drop_na()
# Compute total number of mutations per cancer types
ICGC.snvs.count.type.df <- ICGC.snvs.closest.ori.type.df %>% group_by(cancer.type) %>% summarise(snvs.count=n())
# Compute mutation rates per cancer types
ICGC.snvs.ori.mut.rate.type.df <- ICGC.snvs.closest.ori.type.df %>% mutate(dist = -V14) %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
mutate(mut.type = ifelse((V4 == "G" | V4 == "C"), "GC", "AT")) %>% group_by(cancer.type, dist) %>%
summarise(GC.mut = sum(mut.type == "GC"), AT.mut = sum(mut.type == "AT")) %>%
right_join(ori.bc.summary, by = "dist") %>%
mutate(GC.rate = GC.mut/(G+C), AT.rate=AT.mut/(A+T), mut.rate = GC.rate+AT.rate) %>%
mutate(mut.rate.fold=log2(mut.rate/mean(mut.rate[c(1:20,180:200)])))
# Retained 20 cancer types
# Assess variation of mutation rate at the center of origins
ICGC.snvs.summary.type.df <- ICGC.snvs.ori.mut.rate.type.df %>% filter(dist == 0)
# Define mutation rate quantiles
ICGC.snvs.summary.type.df$MUT <- ntile(ICGC.snvs.summary.type.df$mut.rate.fold, 2)
ICGC.snvs.summary.type.df$rank <- rank(ICGC.snvs.summary.type.df$mut.rate.fold)
# Define groups of low, medium and high mutation rates
type.low.df <- ICGC.snvs.summary.type.df %>% filter(MUT == 1)
type.high.df <- ICGC.snvs.summary.type.df %>% filter(MUT == 2)
# Prepare df
ICGC.snvs.ori.low.mut.rate.type.df <- ICGC.snvs.ori.mut.rate.type.df %>% filter(cancer.type %in% type.low.df$cancer.type)
ICGC.snvs.ori.high.mut.rate.type.df <- ICGC.snvs.ori.mut.rate.type.df %>% filter(cancer.type %in% type.high.df$cancer.type)
# Save df
saveRDS(ICGC.snvs.ori.mut.rate.type.df, "./001_Mut_density_Pan_cancer/rds/ICGC.snvs.ori.mut.rate.type.df.rds")
saveRDS(ICGC.snvs.summary.type.df, "./001_Mut_density_Pan_cancer/rds/ICGC.snvs.summary.type.df.rds")
saveRDS(ICGC.snvs.ori.low.mut.rate.type.df, "./001_Mut_density_Pan_cancer/rds/ICGC.snvs.ori.low.mut.rate.type.df.rds")
saveRDS(ICGC.snvs.ori.high.mut.rate.type.df, "./001_Mut_density_Pan_cancer/rds/ICGC.snvs.ori.high.mut.rate.type.df.rds")Plot results
library(dplyr)
library(tidyr)
library(ggplot2)
library(ggpubr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load df
ICGC.snvs.summary.type.df <- readRDS("./001_Mut_density_Pan_cancer/rds/ICGC.snvs.summary.type.df.rds")
ICGC.snvs.ori.low.mut.rate.type.df <- readRDS("./001_Mut_density_Pan_cancer/rds/ICGC.snvs.ori.low.mut.rate.type.df.rds")
ICGC.snvs.ori.high.mut.rate.type.df <- readRDS("./001_Mut_density_Pan_cancer/rds/ICGC.snvs.ori.high.mut.rate.type.df.rds")
# Define plotting colors
colfunc <- colorRampPalette(c("#29235C", "#309B3E", "#FFDE00", "#BF0808"))
# show_col(colfunc(20))
colfunc.df <- cbind.data.frame(rank = c(1:20), color = colfunc(20)) %>% left_join((ICGC.snvs.summary.type.df %>% dplyr::select(cancer.type, rank))) %>% dplyr::select(cancer.type, color)
# Plot cancers with high mutation rates
ICGC.snvs.ori.high.mut.rate.type.df.2 <- ICGC.snvs.ori.high.mut.rate.type.df %>% left_join(colfunc.df, by = "cancer.type")
ICGC.snvs.ori.high.mut.rate.type.plot <- ICGC.snvs.ori.high.mut.rate.type.df.2 %>%
ggplot(aes(x=dist, y=mut.rate.fold)) + geom_line(aes(color=color)) +
scale_color_identity(name = "Cancer type", breaks = ICGC.snvs.ori.high.mut.rate.type.df.2$color, labels = ICGC.snvs.ori.high.mut.rate.type.df.2$cancer.type, guide = "legend") +
xlim(-5000,5000) + ylim(-1.5,2.0) +
xlab("Distance from origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins") +
geom_hline(yintercept=0, linetype="dashed") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.snvs.ori.high.mut.rate.type.plot
# Plot cancers with low mutation rates
ICGC.snvs.ori.low.mut.rate.type.df.2 <- ICGC.snvs.ori.low.mut.rate.type.df %>% left_join(colfunc.df, by = "cancer.type")
ICGC.snvs.ori.low.mut.rate.type.plot <- ICGC.snvs.ori.low.mut.rate.type.df.2 %>%
ggplot(aes(x=dist, y=mut.rate.fold)) + geom_line(aes(color=color)) +
scale_color_identity(name = "Cancer type", breaks = ICGC.snvs.ori.low.mut.rate.type.df.2$color, labels = ICGC.snvs.ori.low.mut.rate.type.df.2$cancer.type, guide = "legend") +
xlim(-5000,5000) + ylim(-1.5,2.0) +
xlab("Distance from origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins") +
geom_hline(yintercept=0, linetype="dashed") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
ICGC.snvs.ori.low.mut.rate.type.plot
pdf("./Rplots/ICGC.snvs.ori.mut.rate.type.plot.pdf", width=10, height=6, useDingbats=FALSE)
ggarrange(ICGC.snvs.ori.high.mut.rate.type.plot, ICGC.snvs.ori.low.mut.rate.type.plot, ncol = 2)
dev.off()## quartz_off_screen
## 24 Characterisation of structural variations
4.1 Correlations between SVs and snvs burden at origins
The density of SVs at origins (+- 500bp) for genomes with more than 5,000 snvs is computed
# r
library(tidyr)
# Load snvs density ratio df
snvs.dens.ratio.df <- readRDS("./001_Mut_density_Pan_cancer/rds/snvs.dens.ratio.df.rds") %>% distinct()
# Open ori and flank coordinates
ori.1kb.domains.gr <- import("./Dataset/ori/BED/ori.1kb.hg19.bed")
ori.flanks.gr <- import("./Dataset/ori/BED/ori.flanks.hg19.bed")
# Load SV manisfest
SV.manifest.df <- read.table(gzfile("./Dataset/ICGC/SV/manifest.1698320954196.tar.gz"), skip = 1)
SV.manifest.df <- SV.manifest.df %>% dplyr::select(V9, V10, V2, V3, V5)
colnames(SV.manifest.df) <- c("donor.id", "project", "file.id", "object.id", "file.name")
# List SV vcf files
vcf.SV.files <- list.files("./Dataset/ICGC/SV/VCF/") # 3,822 files
vcf.SV.files <- vcf.SV.files[!grepl(".idx", vcf.SV.files)] # 1,911 SV vcf files
vcf.SV.filter.files <- vcf.SV.files[vcf.SV.files %in% SV.manifest.df$file.name] # 1,911 SV vcf files, CHECK OK
# Compute SV counts
SV.dens.ratio.df <- tibble()
for (i in 1:length(vcf.SV.filter.files)) {
vcf.SV.files.i <- vcf.SV.filter.files[i]
print(vcf.SV.files.i)
df.i <- SV.manifest.df %>% filter(file.name == vcf.SV.files.i)
donor.i <- df.i$donor.id
path.i <- paste("./Dataset/ICGC/SV/VCF/", vcf.SV.files.i, sep = "")
vcf.i <- read.table(gzfile(path.i), comment.char = "#") %>% dplyr::select(V1, V2)
count.i <- nrow(vcf.i)
bed.i <- vcf.i %>% mutate(end = V2 + 1) %>% dplyr::select(seqnames = V1, start = V2, end)
gr.i <- makeGRangesFromDataFrame(bed.i)
ori.SV.overlap.i <- subsetByOverlaps(gr.i, ori.1kb.domains.gr)
SV.ori.count.i <- (length(ori.SV.overlap.i)*1000)/9340000
flanks.SV.overlap.i <- subsetByOverlaps(gr.i, ori.flanks.gr)
SV.flanks.count.i <- (length(flanks.SV.overlap.i)*1000)/177441320
SV.df.summary.i <- cbind.data.frame(donor = donor.i, SV.count = count.i, SV.dens.ori = SV.ori.count.i, SV.dens.flanks = SV.flanks.count.i) %>% mutate(SV.dens.ratio = SV.dens.ori/SV.dens.flanks)
SV.dens.ratio.df <- rbind(SV.dens.ratio.df,SV.df.summary.i)
}
plot(log10(SV.dens.ratio.df$SV.count), SV.dens.ratio.df$SV.dens.ratio)
saveRDS(SV.dens.ratio.df, "./001_Mut_density_Pan_cancer/rds/SV.dens.ratio.df.rds")
# Add SV information to snvs density ratio information
snvs.SV.dens.ratio.df <- snvs.dens.ratio.df %>% distinct() %>% full_join(SV.dens.ratio.df, by = "donor") %>% distinct()
saveRDS(snvs.SV.dens.ratio.df, "./001_Mut_density_Pan_cancer/rds/snvs.SV.dens.ratio.df.rds")Analyse results
library(dplyr)
library(tidyr)
library(ggplot2)
library(scales)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load snvs density ratio df
snvs.SV.dens.ratio.df <- readRDS("./001_Mut_density_Pan_cancer/rds/snvs.SV.dens.ratio.df.rds") %>% drop_na()
# Assess the distribution of SV density stratified by primary sites
snvs.SV.dens.ratio.plot <- snvs.SV.dens.ratio.df %>%
ggplot(aes(x=reorder(Primary.Site,-log1p(SV.dens.ratio), na.rm=T), y=log1p(SV.dens.ratio), fill="#E41F1A", alpha = 0.3)) +
geom_boxplot(outlier.shape = NA, width = 0.5) +
geom_jitter(color="black", size=0.4, alpha=0.4) + ylab("SV density ratio (ori/flanks, log1p scale)") +
geom_hline(yintercept=0.69, linetype="dashed") +
theme_bw() + theme(aspect.ratio=0.4, panel.grid.minor = element_line(linetype = "dotted"), legend.position = "none", axis.title.x=element_blank(), axis.text.x=element_text(angle=45,hjust=1))
snvs.SV.dens.ratio.plot
# Assess the correlation between snvs and SV density ratio
snvs.SV.dens.ratio.clean.df <- snvs.SV.dens.ratio.df %>% filter(snvs.dens.ratio > 0 & SV.dens.ratio > 0 & SV.dens.ratio != "Inf")
snvs.SV.dens.ratio.plot.2 <- snvs.SV.dens.ratio.clean.df %>%
ggplot(aes(x=log1p(snvs.dens.ratio), y=log1p(SV.dens.ratio))) +
geom_point(alpha = 0.2) +
geom_smooth(method= "lm", se = T) +
geom_hline(yintercept=0.69, linetype="dashed") + geom_vline(xintercept=0.69, linetype="dashed") +
xlab("Mutation density ratio (ori/flanks, log1p scale)") + ylab("SV density ratio (ori/flanks, log1p scale)") + ggtitle("Rho = 0.2592406, Pval < 2.2e-16") +
theme_bw() + theme(aspect.ratio=1, panel.grid.minor = element_line(linetype = "dotted"))
snvs.SV.dens.ratio.plot.2
cor.test(snvs.SV.dens.ratio.clean.df$snvs.dens.ratio, snvs.SV.dens.ratio.clean.df$SV.dens.ratio)##
## Pearson's product-moment correlation
##
## data: snvs.SV.dens.ratio.clean.df$snvs.dens.ratio and snvs.SV.dens.ratio.clean.df$SV.dens.ratio
## t = 10.69, df = 1586, p-value < 2.2e-16
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
## 0.2127631 0.3045476
## sample estimates:
## cor
## 0.2592406# Rho = 0.2592406, Pval < 2.2e-16
pdf("./Rplots/SV.snvs.cor.pancancer.pdf", width=7, height=6, useDingbats=FALSE)
snvs.SV.dens.ratio.plot.2
dev.off()## quartz_off_screen
## 24.2 Origin-associated SVs characterisation
Convert SV vcf into annotated gr objects
# r
source("./Scripts/SV_annotation.R")
library(StructuralVariantAnnotation)
# Load SV manisfest
SV.manifest.df <- read.table(gzfile("./Dataset/ICGC/SV/manifest.1698320954196.tar.gz"), skip = 1)
SV.manifest.df <- SV.manifest.df %>% dplyr::select(V9, V10, V2, V3, V5)
colnames(SV.manifest.df) <- c("donor.id", "project", "file.id", "object.id", "file.name")
# List SV vcf files
vcf.SV.files <- list.files("./Dataset/ICGC/SV/VCF/") # 3,822 files
vcf.SV.files <- vcf.SV.files[!grepl(".idx", vcf.SV.files)] # 1,911 SV vcf files
vcf.SV.filter.files <- vcf.SV.files[vcf.SV.files %in% SV.manifest.df$file.name] # 1,911 SV vcf files, CHECK OK
# Annotate and save gr objects
for (i in 1:length(vcf.SV.filter.files)) {
vcf.SV.files.i <- vcf.SV.filter.files[i]
print(i)
df.i <- SV.manifest.df %>% filter(file.name == vcf.SV.files.i)
donor.i <- df.i$donor.id
path.i <- paste("./Dataset/ICGC/SV/VCF/", vcf.SV.files.i, sep = "")
vcf.i <- readVcf(path.i, "hg19")
gr.i <- breakpointRanges(vcf.i)
event.gr.i <- simpleEventType(gr.i)
gr.i$svtype <- event.gr.i
path.out.i <- paste0("./Dataset/ICGC/SV/annoGR/", donor.i, ".annoSV.gr.rds")
saveRDS(gr.i, path.out.i)
}Compute the distribution of SV types at the vicinity of replication origins
# r
# Load origin coordinates
ori.center.df <- read.table("./Dataset/ori/ori.inter.hg19.NCBI.bed") %>% dplyr::select(V1, V2, V3)
colnames(ori.center.df) <- c("seqnames", "start", "end")
ori.center.gr <- makeGRangesFromDataFrame(ori.center.df)
# List SV annotated gr files
annoGR.SV.files <- list.files("./Dataset/ICGC/SV/annoGR/") # 1,794 files
annoGR.SV.donor <- str_replace(annoGR.SV.files, ".annoSV.gr.rds", "")
path.SV.files <- paste0("./Dataset/ICGC/SV/annoGR/", annoGR.SV.files)
# Compute distances from each SV to the closest origins
# SV at more than +-10 kb of an origin are filtered out
annoSV.ori.dist.df <- tibble()
for (i in 1:length(annoGR.SV.files)) {
print(i/length(annoGR.SV.files))
gr.i <- readRDS(path.SV.files[i])
donor.i <- annoGR.SV.donor[i]
nearest.i <- nearest(gr.i, ori.center.gr)
nearest.ori.i <- start(ori.center.gr[nearest.i])
df.i <- as.data.frame(gr.i) %>% dplyr::select(seqnames, start, end, REF, ALT, svtype, svLen, insLen)
df.i <- df.i %>% mutate(nearest.ori.i = nearest.ori.i, dist.ori = start - nearest.ori.i, donor = donor.i) %>%
filter(dist.ori >= -10000 & dist.ori <= 10000) %>% dplyr::select(-nearest.ori.i)
rownames(df.i) <- NULL
annoSV.ori.dist.df <- rbind(annoSV.ori.dist.df, df.i)
}
saveRDS(annoSV.ori.dist.df, "./001_Mut_density_Pan_cancer/rds/annoSV.ori.dist.df.rds")
# 83,659 SVsAnalyse origin-associated SV
library(dplyr)
library(tidyr)
library(ggplot2)
library(forcats)
library(ggpubr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load results
annoSV.ori.dist.df <- readRDS("./001_Mut_density_Pan_cancer/rds/annoSV.ori.dist.df.rds")
# Plot the distribution of small SV type in the vicinity of origins
# Compute the distances in bins of 100 nt
annoSV.ori.dist.summary.df <- annoSV.ori.dist.df %>% mutate(dist = (as.numeric(cut(dist.ori, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
group_by(dist, svtype) %>% summarise(count = n()) %>% group_by(dist) %>% mutate(freq=count/sum(count))
annoSV.ori.dist.summary.plot <- annoSV.ori.dist.summary.df %>% mutate(SV = fct_relevel(svtype, "INV", "DUP", "DEL", "ITX", "INS")) %>%
ggplot(aes(x = dist, y = freq)) +
geom_col(aes(fill = SV), width = 100, alpha = 0.8) + xlim(-5000,5000) +
scale_fill_manual(values = c("#D4D2D2", "#BF0808", "#4F4A75", "#EAA813", "#94C994")) +
xlab("Distance from origin (bp)") + ylab("Relative contribution") +
theme_classic() + theme(aspect.ratio=1)
annoSV.ori.dist.summary.plot
# Tandem duplication characterisation
# Recover duplication at origins and flanks
SD.ori.df <- annoSV.ori.dist.df %>% filter(dist.ori >= -500 & dist.ori <= 500) %>% filter(svtype == "DUP") %>% mutate(class = "ori") # 1,445 SVs
SD.flanks.df <- annoSV.ori.dist.df %>% filter(dist.ori < -500 | dist.ori > 500) %>% filter(svtype == "DUP") %>% mutate(class = "flanks") # 12,295 SVs
SD.summary.df <- rbind(SD.ori.df, SD.flanks.df)
SD.summary.plot <- SD.summary.df %>%
ggplot(aes(x=class, y=svLen, alpha = 0.3)) +
geom_boxplot(width = 0.5, fill="#D56114") +
scale_y_continuous(trans = log10_trans(), limits=c(10,100000)) + ylab("Tandem duplication length (bb)") + ggtitle("Pval < 2.2e-16") +
theme_bw() + theme(aspect.ratio=2, panel.grid.minor = element_line(linetype = "dotted"), legend.position = "none", axis.title.x=element_blank(), axis.text.x=element_text(angle=45,hjust=1))
SD.summary.plot
# Compute stats
ks.test(SD.ori.df$svLen, SD.flanks.df$svLen) # p-value < 2.2e-16##
## Asymptotic two-sample Kolmogorov-Smirnov test
##
## data: SD.ori.df$svLen and SD.flanks.df$svLen
## D = 0.36765, p-value < 2.2e-16
## alternative hypothesis: two-sided# Save plot
pdf("./Rplots/SV.annotation.pancancer.pdf", width=12, height=6, useDingbats=FALSE)
ggarrange(annoSV.ori.dist.summary.plot, SD.summary.plot, nrow = 1)
dev.off()## quartz_off_screen
## 24.3 SV signatures
SV signatures are computed following the nomenclature introduced in Li et al. Nature 2020, 578, 112-121 (https://www.nature.com/articles/s41586-019-1913-9). We consider each type of SV binned in 8 length intervals. Interchromosomal/translocation are counted without considering segment length.
# r
# Load results
annoSV.ori.dist.df <- readRDS("./001_Mut_density_Pan_cancer/rds/annoSV.ori.dist.df.rds")
unique(annoSV.ori.dist.df$svtype)
# "ITX" "DUP" "INV" "DEL" "INS"
# Define length ranges
SV.sig.df <- annoSV.ori.dist.df %>%
mutate(svlength = case_when(svtype != "ITX" & abs(svLen) >= 0 & abs(svLen) <= 100 ~ "0-0.1kb",
svtype != "ITX" & abs(svLen) > 100 & abs(svLen) <= 500 ~ "0.1-0.5kb",
svtype != "ITX" & abs(svLen) > 500 & abs(svLen) <= 1000 ~ "0.5-1kb",
svtype != "ITX" & abs(svLen) > 1000 & abs(svLen) <= 5000 ~ "1-5kb",
svtype != "ITX" & abs(svLen) > 5000 & abs(svLen) <= 10000 ~ "5-10kb",
svtype != "ITX" & abs(svLen) > 10000 & abs(svLen) <= 100000 ~ "10-100kb",
svtype != "ITX" & abs(svLen) > 100000 & abs(svLen) <= 1000000 ~ "0.1-1Mb",
svtype != "ITX" & abs(svLen) > 1000000 ~ ">1Mb",
svtype == "ITX" ~ "ITX",
T ~ NA)) %>% filter(svlength != "NA")
# Prepare empty table
intervals <- c("0-0.1kb", "0.1-0.5kb", "0.5-1kb", "1-5kb", "5-10kb", "10-100kb", "0.1-1Mb", ">1Mb")
SV.sig.init.df <- expand.grid(c("INS", "DEL", "DUP", "INV"), intervals)
colnames(SV.sig.init.df) <- c("svtype", "svlength")
SV.sig.init.df <- rbind(SV.sig.init.df, cbind(svtype = "ITX", svlength = "ITX"))
# Recover origin associated signatures
SV.sig.ori.df <- SV.sig.df %>% filter(dist.ori >= -500 & dist.ori <= 500) %>% group_by(svtype, svlength) %>% summarise(count = n()) %>%
right_join(SV.sig.init.df, by = c("svtype", "svlength")) %>% ungroup() %>% mutate(freq = count/sum(count, na.rm = T)) %>% select(-count)
SV.sig.ori.df[is.na(SV.sig.ori.df)] <- 0
saveRDS(SV.sig.ori.df, "./001_Mut_density_Pan_cancer/rds/SV.sig.ori.df.rds")
# Recover flanks associated signatures
SV.sig.flanks.df <- SV.sig.df %>% filter(dist.ori <= -500 | dist.ori >= 500) %>% group_by(svtype, svlength) %>% summarise(count = n()) %>%
right_join(SV.sig.init.df, by = c("svtype", "svlength")) %>% ungroup() %>% mutate(freq = count/sum(count, na.rm = T)) %>% select(-count)
SV.sig.flanks.df[is.na(SV.sig.flanks.df)] <- 0
saveRDS(SV.sig.flanks.df, "./001_Mut_density_Pan_cancer/rds/SV.sig.flanks.df.rds")Plot
library(dplyr)
library(tidyr)
library(ggplot2)
library(forcats)
library(ggpubr)
setwd("/Users/pm23/Desktop/Projects/OriCan")
# Load results
SV.sig.ori.df <- readRDS("./001_Mut_density_Pan_cancer/rds/SV.sig.ori.df.rds")
SV.sig.flanks.df <- readRDS("./001_Mut_density_Pan_cancer/rds/SV.sig.flanks.df.rds")
# Plot
SV.sig.ori.plot <- SV.sig.ori.df %>%
mutate(SVlength = fct_relevel(svlength, "0-0.1kb", "0.1-0.5kb", "0.5-1kb", "1-5kb", "5-10kb", "10-100kb", "0.1-1Mb", ">1Mb")) %>%
mutate(SVtype = fct_relevel(svtype, "INS", "DEL", "DUP", "INV", "ITX")) %>%
ggplot(aes(x = SVlength, y = freq, fill = SVtype, width = 1)) +
geom_bar(stat = "identity", colour = "black", size = .2) + ylim(0,0.17) + ylab("Probability") + xlab("SV types") +
scale_fill_manual(values = c("#94C994", "#4F4A75", "#BF0808", "#D4D2D2", "#EAA813")) +
theme_bw() + facet_grid(.~SVtype, space = "free", scales = "free_x")
SV.sig.flanks.plot <- SV.sig.flanks.df %>%
mutate(SVlength = fct_relevel(svlength, "0-0.1kb", "0.1-0.5kb", "0.5-1kb", "1-5kb", "5-10kb", "10-100kb", "0.1-1Mb", ">1Mb")) %>%
mutate(SVtype = fct_relevel(svtype, "INS", "DEL", "DUP", "INV", "ITX")) %>%
ggplot(aes(x = SVlength, y = freq, fill = SVtype, width = 1)) +
geom_bar(stat = "identity", colour = "black", size = .2) + ylim(0,0.17) + ylab("Probability") +
scale_fill_manual(values = c("#94C994", "#4F4A75", "#BF0808", "#D4D2D2", "#EAA813")) +
theme_bw() + facet_grid(.~SVtype, space = "free", scales = "free_x")
ggarrange(SV.sig.ori.plot, SV.sig.flanks.plot, nrow = 2)
# Save plot
pdf("./Rplots/SV.signatures.pdf", width=6, height=6, useDingbats=FALSE)
ggarrange(SV.sig.ori.plot, SV.sig.flanks.plot, nrow = 2)
dev.off()## quartz_off_screen
## 25 Mutational burden at origin in non-cancerous cells
Analyse data from Abascal et al. Nature 2021 (https://www.nature.com/articles/s41586-021-03477-4) and Moore et al. Nature 2021 (https://www.nature.com/articles/s41586-021-03822-7#MOESM4)
# R
###########################################################################
# Analyse data from Abascal et al. Nature 2021 (https://www.nature.com/articles/s41586-021-03477-4)
# data is hg19
# Load supporting information table
library(readxl)
Abascal.ESI.df <- read_excel("./Dataset/Abascal_Nature_2021/41586_2021_3477_MOESM3_ESM.xlsx", sheet = 5)
# Format snvs data
Nanoseq.snvs.df <- Abascal.ESI.df %>% dplyr::select(seqnames= chr, start = pos, end = pos, REF = ref, ALT = mut, sample.id = sampleID)
Nanoseq.snvs.gr <- makeGRangesFromDataFrame(Nanoseq.snvs.df, keep.extra.columns = T)
# Compute distance to origins
ori.df <- read.table("./Dataset/ori/ori.inter.hg19.NCBI.bed", sep="\t")
colnames(ori.df) <- c("seqnames", "start", "end", "EFF", "ori.id", "ori.width")
ori.gr <- makeGRangesFromDataFrame(ori.df, keep.extra.columns = T)
Nanoseq.snvs.ori.nearest <- nearest(Nanoseq.snvs.gr, ori.gr)
ori.nearest.df <- ori.df[Nanoseq.snvs.ori.nearest,]
Nanoseq.snvs.ori.dist.df <- cbind.data.frame(Nanoseq.snvs.df, ori.pos = ori.nearest.df$start) %>% mutate(dist = start-ori.pos) %>%
filter(dist >= -10000 & dist <= 10000) %>% mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100)
# Define tissue type
Nanoseq.snvs.ori.dist.df <- Nanoseq.snvs.ori.dist.df %>%
mutate(tissue = case_when(grepl("grans", sample.id) == T ~ "grans",
grepl("cordblood", sample.id) == T ~ "cord blood",
grepl("smoothmuscle", sample.id) == T ~ "smooth muscle",
grepl("neurons", sample.id) == T ~ "neurons",
grepl("coloniccrypt", sample.id) == T ~ "colonic crypt",
grepl("sperm", sample.id) == T ~ "sperm"))
saveRDS(Nanoseq.snvs.ori.dist.df, "./001_Mut_density_Pan_cancer/rds/Nanoseq.snvs.ori.dist.df.rds")
# Mutation rates by pyrimidine mutation types
Nanoseq.mut.dist.df <- Nanoseq.snvs.ori.dist.df %>% mutate(mut = paste(REF, ALT, sep = ">")) %>%
mutate(pyr = case_when(mut == "A>C" ~ "T>G",
mut == "A>G" ~ "T>C",
mut == "A>T" ~ "T>A",
mut == "G>A" ~ "C>T",
mut == "G>C" ~ "C>G",
mut == "G>T" ~ "C>A",
T ~ mut)) %>% group_by(pyr, dist) %>% summarise(pyr.rate = n()) %>%
mutate(pyr.rate.cor = log2(pyr.rate/mean(pyr.rate[c(1:20,180:200)], na.rm = T)))
saveRDS(Nanoseq.mut.dist.df, "./001_Mut_density_Pan_cancer/rds/Nanoseq.mut.dist.df.rds")
###########################################################################
# Analyse data from Moore et al. Nature 2021 (https://www.nature.com/articles/s41586-021-03822-7#MOESM4)
# data is hg19
Moore.ESI.df <- read.csv("./Dataset/Moore_Nature_2021/Supplementary_Table5.txt", sep = "\t")
# Format snvs data
Soma.snvs.df <- Moore.ESI.df %>% dplyr::select(seqnames= Chr, start = Start, end = End, REF = Ref, ALT = Alt, sample.id = Sample)
Soma.snvs.gr <- makeGRangesFromDataFrame(Soma.snvs.df, keep.extra.columns = T)
# Compute distance to origins
Soma.snvs.ori.nearest <- nearest(Soma.snvs.gr, ori.gr)
ori.nearest.df <- ori.df[Soma.snvs.ori.nearest,]
Soma.snvs.ori.dist.df <- cbind.data.frame(Soma.snvs.df, ori.pos = ori.nearest.df$start) %>% mutate(dist = start-ori.pos) %>%
filter(dist >= -10000 & dist <= 10000) %>% mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100)
saveRDS(Soma.snvs.ori.dist.df, "./001_Mut_density_Pan_cancer/rds/Soma.snvs.ori.dist.df.rds")
# Pyrimidine mutation rates
# Load base composition statistics
ori.bc.summary <- read.csv("./Dataset/ori/ori.bc.summary.csv")
# For GC/AT mutations
Soma.snvs.ori.mut.rate.df <- Soma.snvs.ori.dist.df %>%
mutate(mut.type = ifelse((REF == "G" | REF == "C"), "GC", "AT")) %>% group_by(dist) %>%
summarise(GC.mut = sum(mut.type == "GC"), AT.mut = sum(mut.type == "AT")) %>%
right_join(ori.bc.summary) %>%
mutate(GC.rate = GC.mut/(G+C), AT.rate=AT.mut/(A+T)) %>%
mutate(GC.rate.fold=log2(GC.rate/mean(GC.rate[c(1:20,180:200)], na.rm = T)), AT.rate.fold=log2(AT.rate/mean(AT.rate[c(1:20,180:200)], na.rm = T)))
Soma.GC.snvs.ori.mut.rate.df <- Soma.snvs.ori.mut.rate.df %>% dplyr::select(dist, rate = GC.rate.fold) %>% mutate(type = "GC")
Soma.AT.snvs.ori.mut.rate.df <- Soma.snvs.ori.mut.rate.df %>% dplyr::select(dist, rate = AT.rate.fold) %>% mutate(type = "AT")
# For each pyrimidine mutations
# C>A
Soma.CA.ori.mut.rate.df <- Soma.snvs.ori.dist.df %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((REF == "C" & ALT == "A")|(REF == "G" | ALT == "T")) %>%
group_by(dist) %>% summarise(CA.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(CA.rate=CA.mut/(G+C), CA.rate.fold=log2(CA.rate/mean(CA.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CA.rate.fold) %>% mutate(type = "C>A")
# C>G
Soma.CG.ori.mut.rate.df <- Soma.snvs.ori.dist.df %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((REF == "C" & ALT == "G")|(REF == "G" | ALT == "C")) %>%
group_by(dist) %>% summarise(CG.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(CG.rate=CG.mut/(G+C), CG.rate.fold=log2(CG.rate/mean(CG.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CG.rate.fold) %>% mutate(type = "C>G")
# C>T
Soma.CT.ori.mut.rate.df <- Soma.snvs.ori.dist.df %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((REF == "C" & ALT == "T")|(REF == "G" | ALT == "A")) %>%
group_by(dist) %>% summarise(CT.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(CT.rate=CT.mut/(G+C), CT.rate.fold=log2(CT.rate/mean(CT.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = CT.rate.fold) %>% mutate(type = "C>T")
# T>A
Soma.TA.ori.mut.rate.df <- Soma.snvs.ori.dist.df %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((REF == "T" & ALT == "A")|(REF == "A" | ALT == "T")) %>%
group_by(dist) %>% summarise(TA.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(TA.rate=TA.mut/(T+A), TA.rate.fold=log2(TA.rate/mean(TA.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TA.rate.fold) %>% mutate(type = "T>A")
# T>C
Soma.TC.ori.mut.rate.df <- Soma.snvs.ori.dist.df %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((REF == "T" & ALT == "C")|(REF == "A" | ALT == "G")) %>%
group_by(dist) %>% summarise(TC.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(TC.rate=TC.mut/(T+A), TC.rate.fold=log2(TC.rate/mean(TC.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TC.rate.fold) %>% mutate(type = "T>C")
# T>G
Soma.TG.ori.mut.rate.df <- Soma.snvs.ori.dist.df %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
filter((REF == "T" & ALT == "G")|(REF == "A" | ALT == "C")) %>%
group_by(dist) %>% summarise(TG.mut=n()) %>% right_join(ori.bc.summary) %>% mutate(TG.rate=TG.mut/(T+A), TG.rate.fold=log2(TG.rate/mean(TG.rate[c(1:20,180:200)]))) %>% dplyr::select(dist, rate = TG.rate.fold) %>% mutate(type = "T>G")
# Prepare summary df
Soma.snvs.ori.mut.summary.rate.df <- rbind(Soma.GC.snvs.ori.mut.rate.df, Soma.AT.snvs.ori.mut.rate.df,
Soma.CA.ori.mut.rate.df, Soma.CG.ori.mut.rate.df, Soma.CT.ori.mut.rate.df,
Soma.TA.ori.mut.rate.df, Soma.TC.ori.mut.rate.df, Soma.TG.ori.mut.rate.df)
saveRDS(Soma.snvs.ori.mut.summary.rate.df, "./001_Mut_density_Pan_cancer/rds/Soma.snvs.ori.mut.summary.rate.df.rds")
# Compute mutation rates per somatic tissue
# List tissue information
Moore.tissue.info.df <- read_excel("./Dataset/Moore_Nature_2021/41586_2021_3822_MOESM4_ESM.xlsx", sheet = 4, col_names = TRUE)
colnames(Moore.tissue.info.df) <- Moore.tissue.info.df[1,]
Moore.tissue.info.df <- Moore.tissue.info.df[-1,]
Moore.tissue.info.format.df <- Moore.tissue.info.df %>% dplyr::select(sample.id = Sample, tissue = TissueType1)
Soma.tissue.rate.df <- Soma.snvs.ori.dist.df %>% left_join(Moore.tissue.info.format.df, by = "sample.id")
Soma.tissue.rate.df <- Soma.tissue.rate.df %>%
mutate(dist = (as.numeric(cut(dist, breaks = seq(-10000, 10000, 100)))-100)*100) %>%
mutate(mut.type = ifelse((REF == "G" | REF == "C"), "GC", "AT")) %>%
group_by(tissue, dist) %>%
summarise(GC.mut = sum(mut.type == "GC"), AT.mut = sum(mut.type == "AT")) %>%
right_join(ori.bc.summary, by = "dist") %>%
mutate(GC.rate = GC.mut/(G+C), AT.rate=AT.mut/(A+T), mut.rate = GC.rate+AT.rate) %>%
mutate(mut.rate.fold=log2(mut.rate/mean(mut.rate[c(1:20,180:200)])))
saveRDS(Soma.tissue.rate.df, "./001_Mut_density_Pan_cancer/rds/Soma.tissue.rate.df.rds")Plot
library(dplyr)
library(tidyr)
library(ggplot2)
library(forcats)
library(ggpubr)
library(wesanderson)
setwd("/Users/pm23/Desktop/Projects/OriCan")
###########################################################################
# Data from Abascal et al. Nature 2021 (https://www.nature.com/articles/s41586-021-03477-4)
# Load results
Nanoseq.snvs.ori.dist.df <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/Nanoseq.snvs.ori.dist.df.rds")
Nanoseq.mut.dist.df <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/Nanoseq.mut.dist.df.rds")
# Plot
Nanoseq.snvs.ori.count.plot <- Nanoseq.snvs.ori.dist.df %>% group_by(dist) %>% summarise(count = n()) %>%
ggplot(aes(x=dist, y=count)) +
geom_line() + xlim(-5000, 5000) +
xlab("Distance from origin (bp)") + ylab("snvs count") + ggtitle("Nanoseq snvs counts at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
Nanoseq.snvs.ori.count.plot
Nanoseq.pyr.rate.ori.plot <- Nanoseq.mut.dist.df %>% ggplot(aes(x=dist, y=pyr.rate.cor, colour = pyr)) +
geom_line() + xlim(-5000, 5000) + ylim(-3, 3) +
xlab("Distance from origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("Nanoseq snvs mutation rate at origins") +
scale_color_manual(values=c("#E69F00", "#56B4E9", "#009E73","#CC79A7", "#0072B2", "#D55E00")) +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
Nanoseq.pyr.rate.ori.plot
###########################################################################
# Data from Moore et al. Nature 2021 (https://www.nature.com/articles/s41586-021-03822-7#MOESM4)
# Load results
Soma.snvs.ori.dist.df <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/Soma.snvs.ori.dist.df.rds")
Soma.snvs.ori.mut.summary.rate.df <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/Soma.snvs.ori.mut.summary.rate.df.rds")
Soma.tissue.rate.df <- readRDS("/Users/pm23/Desktop/Projects/OriCan/001_Mut_density_Pan_cancer/rds/Soma.tissue.rate.df.rds")
# Plot
Soma.snvs.ori.count.plot <- Soma.snvs.ori.dist.df %>% group_by(dist) %>% summarise(count = n()) %>%
ggplot(aes(x=dist, y=count)) +
geom_line() + xlim(-5000, 5000) + ylim(90, 200) +
xlab("Distance from origin (bp)") + ylab("snvs count") + ggtitle("Non-cancerous snvs count at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
Soma.snvs.ori.count.plot
Soma.pyr.rate.plot <- Soma.snvs.ori.mut.summary.rate.df %>% filter(type != "GC" & type != "AT") %>%
ggplot(aes(x=dist, y=rate, colour=type)) + geom_line() +
scale_color_manual(values=c("#E69F00", "#56B4E9", "#009E73","#CC79A7", "#0072B2", "#D55E00")) +
xlim(-5000,5000) + ylim(-0.5,1.65) +
xlab("Distance from origin (bp)") + ylab("Mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
Soma.pyr.rate.plot
palette <- wes_palette("Zissou1", length(unique(Soma.tissue.rate.df$tissue)), type = "continuous")
Soma.tissue.GC.rate.plot <- Soma.tissue.rate.df %>%
ggplot(aes(x=dist, y=GC.rate, colour=tissue)) + geom_line() +
scale_colour_manual(values=c(palette)) +
xlim(-5000,5000) + #ylim(-0.5,1.65) +
xlab("Distance from origin (bp)") + ylab("GC>AT mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
Soma.tissue.GC.rate.plot
Soma.tissue.AT.rate.plot <- Soma.tissue.rate.df %>%
ggplot(aes(x=dist, y=AT.rate, colour=tissue)) + geom_line() +
scale_colour_manual(values=c(palette)) +
xlim(-5000,5000) + #ylim(-0.5,1.65) +
xlab("Distance from origin (bp)") + ylab("AT>CG mutation rate (log2 FC from background)") + ggtitle("snvs mutation rate at origins") +
theme_bw() + theme(aspect.ratio=1.0, panel.grid.minor = element_line(linetype = "dotted"))
Soma.tissue.AT.rate.plot
###########################################################################
# Combine and save plots
pdf("/Users/pm23/Desktop/Projects/OriCan/Manuscript/Nature Communications/Rplots/Nanoseq_mut_rates.pdf", width=14, height=8, useDingbats=FALSE)
ggarrange(Nanoseq.snvs.ori.count.plot, Nanoseq.pyr.rate.ori.plot,
Soma.snvs.ori.count.plot, Soma.tissue.GC.rate.plot, Soma.tissue.AT.rate.plot, nrow = 2, ncol = 3)
dev.off()## quartz_off_screen
## 2